U.S. patent application number 17/269246 was filed with the patent office on 2022-07-07 for antigen-binding proteins targeting shared antigens.
The applicant listed for this patent is Gritstone bio, Inc.. Invention is credited to Wade Blair, Jennifer Busby, Michele Anne Busby, Gijsbert Marnix Grotenbreg, Karin Jooss, Godfrey Jonah Anderson Rainey, Mojca Skoberne, Roman Yelensky.
Application Number | 20220213196 17/269246 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220213196 |
Kind Code |
A1 |
Jooss; Karin ; et
al. |
July 7, 2022 |
ANTIGEN-BINDING PROTEINS TARGETING SHARED ANTIGENS
Abstract
Provided herein are HLA-PEPTIDE targets and antigen binding
proteins that bind HLA-PEPTIDE targets. Also disclosed are methods
for identifying the HLA-PEPTIDE targets as well as identifying one
or more antigen binding proteins that bind a given HLA-PEPTIDE
target.
Inventors: |
Jooss; Karin; (Emeryville,
CA) ; Rainey; Godfrey Jonah Anderson; (San Diego,
CA) ; Blair; Wade; (Gaithersburg, MD) ; Busby;
Michele Anne; (Needham, MA) ; Busby; Jennifer;
(Burlington, MA) ; Grotenbreg; Gijsbert Marnix;
(Berkeley, CA) ; Skoberne; Mojca; (Cambridge,
MA) ; Yelensky; Roman; (Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gritstone bio, Inc. |
Emeryville |
CA |
US |
|
|
Appl. No.: |
17/269246 |
Filed: |
August 16, 2019 |
PCT Filed: |
August 16, 2019 |
PCT NO: |
PCT/US19/46967 |
371 Date: |
February 17, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62719565 |
Aug 17, 2018 |
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62808775 |
Feb 21, 2019 |
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62869923 |
Jul 2, 2019 |
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International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 14/47 20060101 C07K014/47; C07K 14/74 20060101
C07K014/74; C07K 16/30 20060101 C07K016/30; C12N 15/10 20060101
C12N015/10; C40B 40/08 20060101 C40B040/08; C40B 40/10 20060101
C40B040/10 |
Claims
1. An isolated antigen binding protein (ABP) that specifically
binds to a human leukocyte antigen (HLA)-PEPTIDE target, wherein
the HLA-PEPTIDE target comprises an HLA-restricted peptide
complexed with an HLA Class I molecule, wherein the HLA-restricted
peptide is located in the peptide binding groove of an
.alpha.1/.alpha.2 heterodimer portion of the HLA Class I molecule,
wherein the HLA Class I molecule is HLA subtype B*35:01 (reference
sequence:
MGSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRTEPRAPWIEQEGPE
YWDRNTQIFKTNTQTYRESLRNLRGYYNQSEAGSHIIQRMYGCDLGPDGRLLRGHDQS
AYDGKDYIALNEDLSSWTAADTAAQITQRKWEAARVAEQLRAYLEGLCVEWLRRYLE
NGKETLQRADPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV
ETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLR) and the HLA-restricted
peptide comprises the sequence EVDPIGHVY, and wherein the ABP binds
to any one or more of: a. any one or more of amino acid positions
2-9 of the restricted peptide EVDPIGHVY; b. any one or more of
amino acid positions 50, 54, 55, 57, 61, 62, 74, 81, 82 and 85 of
the .alpha.1 helix of HLA subtype B*35:01; and c. any one or more
of amino acid positions 147 and 148 of the .alpha.2 helix of HLA
subtype B*35:01.
2. (canceled)
3. (canceled)
4. (canceled)
5. The isolated ABP of claim 1, wherein the ABP comprises a CDR-H3
comprising a sequence selected from: CARDGVRYYGMDVW,
CARGVRGYDRSAGYW, CASHDYGDYGEYFQHW, CARVSWYCSSTSCGVNWFDPW,
CAKVNWNDGPYFDYW, CATPTNSGYYGPYYYYGMDVW, CARDVMDVW, CAREGYGMDVW,
CARDNGVGVDYW, CARGIADSGSYYGNGRDYYYGMDVW, CARGDYYFDYW,
CARDGTRYYGMDVW, CARDVVANFDYW, CARGHSSGWYYYYGMDVW, CAKDLGSYGGYYW,
CARSWFGGFNYHYYGMDVW, CARELPIGYGMDVW, and CARGGSYYYYGMDVW.
6. The isolated ABP of claim 1, wherein the ABP comprises a CDR-L3
comprising a sequence selected from: CMQGLQTPITF, CMQALQTPPTF,
CQQAISFPLTF, CQQANSFPLTF, CQQANSFPLTF, CQQSYSTPLTF, CQQTYMMPYTF,
CQQSYITPWTF, CQQSYITPYTF, CQQYYTTPYTF, CQQSYSTPLTF, CMQALQTPLTF,
CQQYGSWPRTF, CQQSYSTPVTF, CMQALQTPYTF, CQQANSFPFTF, CMQALQTPLTF,
and CQQSYSTPLTF.
7. (canceled)
8. (canceled)
9. The isolated ABP of claim 1, wherein the ABP comprises a VH
sequence selected from TABLE-US-00066
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGI
INPRSGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDG
VRYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSHDINWVRQAPGQGLEWMGW
MNPNSGDTGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGV
RGYDRSAGYWGQGTLVIVSS,
EVQLLESGGGLVKPGGSLRLSCAASGFSFSSYWMSWVRQAPGKGLEWISY
ISGDSGYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCASHD
YGDYGEYFQHWGQGTLVTVSS,
EVQLLQSGGGLVQPGGSLRLSCAASGFTFSNSDMNWVRQAPGKGLEWVAY
ISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVS
WYCSSTSCGVNWFDPWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNSDMNWVRQAPGKGLEWVAS
ISSSGGYINYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVN
WNDGPYFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNFGVSWLRQAPGQGLEWMGG
IIPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATPT
NSGYYGPYYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGW
INPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDV MDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSGYLVSWVRQAPGQGLEWMGW
INPNSGGTNTAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREG
YGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYIFRNYPMHWVRQAPGQGLEWMGW
INPDSGGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDN
GVGVDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGW
MNPNIGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGI
ADSGSYYGNGRDYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGW
INPNSGVTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGD
YYFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGW
INPNSGDTKYSQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDG
TRYYGMDVWGQGTTVTVSS,
EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSY
ISSSSSYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDV
VANFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGW
MNPDSGSTGYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGH
SSGWYYYYGMDVWGQGTTVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFTSYSMHWVRQAPGKGLEWVSS
ITSFTNTMYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDL
GSYGGYYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGI
INPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSW
FGGFNYHYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGW
MNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREL
PIGYGMDVWGQGTTVTVSS, and
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IIPIVGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
SYYYYGMDVWGQGTTVTVSS.
10. The isolated ABP of claim 1, wherein the ABP comprises a VL
sequence selected from TABLE-US-00067
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTP ITFGQGTRLEIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSSRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP PTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAISFPLTFGQ STKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYS
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYY
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYMMPYTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYGAS
SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPWTFGQGT KVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPYTFGQ GTKLEIK,
DIVMTQSPDSLAVSLGERATINCKTSQSVLYRPNNENYLAWYQQKPGQPP
KLLIYQASIREPGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTT PYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRFLNWYQQKPGKAPKWYGAS
RPQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGT KVEIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSHRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP LTFGGGTKVEIK,
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYA
ASARASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSWPRTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYGAS
RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPVTFGQGT KVEIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP YTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASEDISNHLNWYQQKPGKAPKLLIYD
ALSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGP GTKVDIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP LTFGQGTKVEIK,
and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKVEIK.
11. (canceled)
12. An isolated antigen binding protein (ABP) that specifically
binds to a human leukocyte antigen (HLA)-PEPTIDE target, wherein
the HLA-PEPTIDE target comprises an HLA-restricted peptide
complexed with an HLA Class I molecule, wherein the HLA-restricted
peptide is located in the peptide binding groove of an
.alpha.1/.alpha.2 heterodimer portion of the HLA Class I molecule,
the HLA Class I molecule is HLA subtype A*01:01 (reference
sequence:
MGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQKMEPRAPWIEQEGPE
YWDQETRNMKAHSQTDRANLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQ
DAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAVHAAEQRRVYLEGRCVDGLRRY
LENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL
VETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLR), and the
HLA-restricted peptide comprises the sequence NTDNNLAVY, and
wherein the ABP binds to any one or more of: a. any one or more of
residues 3-9 of the restricted peptide NTDNNLAVY, b. any one or
more of residues 70-85 of the of the alpha 1 helix of HLA subtype
allele A*01:01, and c. any one or more of residues 140-160 of the
alpha 2 helix of HLA subtype allele A*01:01.
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. The isolated ABP of claim 12, wherein the ABP comprises a
CDR-H3 comprising a sequence selected from: CAATEWLGVW, CARANWLDYW,
CARANWLDYW, CARDWVLDYW, CARGEWLDYW, CARGWELGYW, CARDFVGYDDW,
CARDYGDLDYW, CARGSYGMDVW, CARDGYSGLDVW, CARDSGVGMDVW,
CARDGVAVASDYW, CARGVNVDDFDYW, CARGDYTGNWYFDLW, CARANWLDYW,
CARDQFYGGNSGGHDYW, CAREEDYW, CARGDWFDPW, CARGDWFDPW, CARGEWFDPW,
CARSDWFDPW, CARDSGSYFDYW, CARDYGGYVDYW, CAREGPAALDVW, CARERRSGMDVW,
CARVLQEGMDVW, CASERELPFDIW, CAKGGGGYGMDVW, CAAMGIAVAGGMDVW,
CARNWNLDYW, CATYDDGMDVW, CARGGGGALDYW, CALSGNYYGMDVW,
CARGNPWELRLDYW, and CARDKNYYGMDVW.
19. The isolated ABP of claim 12, wherein the ABP comprises a
CDR-L3 comprising a sequence selected from: CQQSYNTPYTF,
CQQSYSTPYTF, CQQSYSTPYSF, CQQSYSTPFTF, CQQSYGVPYTF, CQQSYSAPYTF,
CQQSYSAPYTF, CQQSYSAPYSF, CQQSYSTPYTF, CQQSYSVPYSF, CQQSYSAPYTF,
CQQSYSVPYSF, CQQSYSTPQTF, CQQLDSYPFTF, CQQSYSSPYTF, CQQSYSTPLTF,
CQQSYSTPYSF, CQQSYSTPYTF, CQQSYSTPYTF, CQQSYSTPFTF, CQQSYSTPTF,
CQQTYAIPLTF, CQQSYSTPYTF, CQQSYIAPFTF, CQQSYSIPLTF, CQQSYSNPTF,
CQQSYSTPYSF, CQQSYSDQWTF, CQQSYLPPYSF, CQQSYSSPYTF, CQQSYTTPWTF,
CQQSYLPPYSF, CQEGITYTF, CQQYYSYPFTF, and CQHYGYSPVTF.
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. The isolated ABP of claim 12, wherein the ABP comprises a VH
sequence selected from TABLE-US-00068
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGMINPSGGGTSY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGNPWELRLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSATISWVRQAPGQGLEWMGWIYPNSGGTVY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAATEWLGVWGQGTTVTVSS,
EVQLLQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNSGGTIS
APNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARANWLDYWGQGTLVTVSS,
EVQLLESGAEVKKPGASVKVSCKASGYTFTTYDLAWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARANWLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKSSGYSFDSYVVNWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDWVLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWMNPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGEWLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGWELGYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTINWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDFVGYDDWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGITWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYGDLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSNYILSWVRQAPGQGLEWMGWINPDSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYSFTRYNMHWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGYSGLDVWGKGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNNGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSGVGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFNNYAFSWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGVAVASDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSSYNMHWVRQAPGQGLEWMGWINGNTGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVNVDDFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGWINPDTGYTRY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYTGNWYFDLWGRGTLVTVS S,
EVQLLESGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPYSGGTNY
AQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARANWLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGYTNY
AQNLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDQFYGGNSGGHDYWGQGTLVTV SS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQGLEWMGWMNPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARE-EDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTINWVRQAPGQGLEWMGWINPNSGGANY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDWFDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLMHWVRQAPGQGLEWMGWISPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDWFDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYVHWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGEWFDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMHWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDWFDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSNYAINWVRQAPGQGLEWMGWISPYSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSGSYFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWIYPNTGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYGGYVDYWGQGTLVTVSS,
EVQLLESGAEVKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWMNPNSGGTKY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGPAALDVWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTLTSHLIHWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERRSGMDVWGQGTTVTVSS,
EVQLLESGAEVKKPGASVKVSCKASGYSFTDYIVHWVRQAPGQGLEWMGWINPYSGGTKY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVLQEGMDVWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSNFLINWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASERELPFDIWGQGTMVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYQMFWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKGGGGYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNSGGTNY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAAMGIAVAGGMDVWGQGTLVTVS S,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYHMHWVRQAPGQGLEWMGWIHPDSGGTSY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNWNLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWMNPNSGNTGY
AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCATYDDGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTVNWVRQAPGQGLEWMGWINPNSGGTKY
AQNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGGGALDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGMINPRDDTTDY
ARDFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCALSGNYYGMDVWGQGTTVTVSS, and
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSQYMHWVRQAPGQGLEWMGRIIPLLGIVNY
AQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDKNYYGMDVWGQGTTVTVSS.
25. The isolated ABP of claim 12, wherein the ABP comprises a VL
sequence selected from TABLE-US-00069
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSYPFTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYAASSLRSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYAASTVQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQDISRWLAWYQQKPGKAPKLLIYAASRLQAGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQTISSWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQTISSWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGVPYTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISNWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSVGNWLAWYQQKPGKAPKWYGASSLQTGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQNIGNWLAWYQQKPGKAPKLLIYAASTLQTGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYGASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSVPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISKWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSVPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQTISNYLNWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPQTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASRDIGRAVGWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQLDSYPFTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPYTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSIGRWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYSFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFAQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKWYGASRLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSVSNWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQTYAIPLTFGGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDIGSWLAWYQQKPGKAPKLLIYATSSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASTLQPGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYIAPFTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASRLESGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTFGGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYGVSSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWVAWYQQKPGKAPKLLIYGASNLESGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSDQWTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYLPPYSFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISNWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTYFTLTISSLQPEDFATYYCQQSYSSPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISHYLNWYQQKPGKAPKWYGASSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPWTFGQGTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYLPPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKWYGASRLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQEGITYTFGQGTKVEIK, and
EIVMTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPGQAPRLLIYGASTRATGIPA
RFSGSGSGTEFTLTISSLQSEDFAVYYCQHYGYSPVTFGQGTKLEIK.
26. (canceled)
27. (canceled)
28. An isolated antigen binding protein (ABP) that specifically
binds to a human leukocyte antigen (HLA)-PEPTIDE target, wherein
the HLA-PEPTIDE target comprises an HLA-restricted peptide
complexed with an HLA Class I molecule, wherein the HLA-restricted
peptide is located in the peptide binding groove of an
.alpha.1/.alpha.2 heterodimer portion of the HLA Class I molecule,
wherein the HLA Class I molecule is HLA subtype A*02:01 (reference
sequence:
MGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPE
YWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYH
QYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRR
YLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDT
ELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLR), and the
HLA-restricted peptide comprises the sequence AIFPGAVPAA, and
wherein the ABP binds to any one or more of: a. any one or more of
amino acid positions 1-6 of the restricted peptide AIFPGAVPAA, b.
any one or more of amino acid positions 46, 49, 55, 61, 74, 76, 77,
78, 81 and 84 of the .alpha.1 helix of HLA subtype A*02:01, c. any
one or more of amino acid positions 45-60, 66, 67, and 73 of the
.alpha.1 helix of HLA subtype A*02:01, d. any one or more of amino
acid positions 138, 145, 147, 152-156, 164, 167 of the .alpha.2
helix of HLA subtype A*02:01, and e. any one or more of any one or
more of amino acid positions 56, 59, 60, 63, 64, 66, 67, 70, 73,
74, 132, 150-153, 155, 156, 158-160, 162-164, 166-168, 170, and 171
of HLA subtype A*02:01.
29.-40. (canceled)
41. The isolated ABP of claim 28, wherein the ABP comprises a
CDR-H3 comprising a sequence selected from: CARDDYGDYVAYFQHW,
CARDLSYYYGMDVW, CARVYDFWSVLSGFDIW, CARVEQGYDIYYYYYMDVW,
CARSYDYGDYLNFDYW, CARASGSGYYYYYGMDVW, CAASTWIQPFDYW,
CASNGNYYGSGSYYNYW, CARAVYYDFWSGPFDYW, CAKGGIYYGSGSYPSW,
CARGLYYMDVW, CARGLYGDYFLYYGMDVW, CARGLLGFGEFLTYGMDVW,
CARDRDSSWTYYYYGMDVW, CARGLYGDYFLYYGMDVW, CARGDYYDSSGYYFPVYFDYW, and
CAKDPFWSGHYYYYGMDVW.
42. The isolated ABP of claim 28, wherein the ABP comprises a
CDR-L3 comprising a sequence selected from: CQQNYNSVTF,
CQQSYNTPWTF, CGQSYSTPPTF, CQQSYSAPYTF, CQQSYSTPPTF, CQQSYSAPYTF,
CQQHNSYPPTF, CQQYSTYPITI, CQQANSFPWTF, CQQSHSTPQTF, CQQSYSTPLTF,
CQQSYSTPLTF, CQQTYSTPWTF, CQQYGSSPYTF, CQQSHSTPLTF, CQQANGFPLTF,
and CQQSYSTPLTF.
43. (canceled)
44. (canceled)
45. The isolated ABP of claim 28, wherein the ABP comprises a VH
sequence selected from: TABLE-US-00070
QVQLVQSGAEVKKPGASVKVSCKASGGTFSRSAITWVRQAPGQGLEWMGW
INPNSGATNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDD
YGDYVAYFQHWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYPFIGQYLHWVRQAPGQGLEWMGI
INPSGDSATYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDL
SYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGW
MNPIGGGTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVY
DFWSVLSGFDIWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSG
INWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVE
QGYDIYYYYYMDVWGKGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTLSSYPINWVRQAPGQGLEWMGW
ISTYSGHADYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSY
DYGDYLNFDYWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSS
ISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAS
GSGYYYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYFMHWVRQAPGQGLEWMGM
VNPSGGSETFAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAAST
WIQPFDYWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFDFSIYSMNWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASNG
NYYGSGSYYNYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTLTTYYMIHWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARA
VYYDFWSGPFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGW
INPYSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKGG
IYYGSGSYPSWGQGTLVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGVSWVRQAPGQGLEWMGW
ISPYSGNTDYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGL
YYMDVWGKGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSNMYLHWVRQAPGQGLEWMGW
INPNTGDTNYAQTFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGL
YGDYFLYYGMDVWGQGTKVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGW
MNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGL
LGFGEFLTYGMDVWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGV
INPSGGSTTYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDR
DSSWTYYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSNYMHWVRQAPGQGLEWMGW
MNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGL
YGDYFLYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSHAISWVRQAPGQGLEWMGV
IIPSGGTSYTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDY
YDSSGYYFPVYFDYWGQGTLVTVSS, and
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGW
INPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDP
FWSGHYYYYGMDVWGQGTTVTVSS.
46. The isolated ABP of claim 28, wherein the ABP comprises a VL
sequence selected from: TABLE-US-00071
DIQMTQSPSSLSASVGDRVTITCRASQSITSYLNWYQQKPGKAPKWYDAS
NLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYNSVTFGQGTK LEIK,
DIQMTQSPSSLSASVGDRVTITCWASQGISSYLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPWTFGP GTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQAISNSLAWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSTPPTFGQ GTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYKAS
SLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGPGT KVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPPTFGG GTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGINSYLAWYQQKPGKAPKWYDAS
NLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNSYPPTFGQGT KLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPITIGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQ GTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQDVSTWLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPQTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYD
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPWTFGQ GTKLEIK,
EIVMTQSPATLSVSPGERATLSCRASQSVGNSLAWYQQKPGQAPRLLIYG
ASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSSPYTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPLTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQNIYTYLNWYQQKPGKAPKWYDAS
NLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGGGT KVEIK, and
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKVEIK.
47.-113. (canceled)
114. A method of treating cancer in a subject, comprising
administering to the subject an effective amount of the antigen
binding protein of claim 1, optionally wherein the cancer is
selected from a solid tumor and a hematological tumor.
115. (canceled)
116. (canceled)
117. (canceled)
118. (canceled)
119. (canceled)
120. A method of treating cancer in a subject, comprising
administering to the subject an effective amount of the antigen
binding protein of claim 12, optionally wherein the cancer is
selected from a solid tumor and a hematological tumor.
121. A method of treating cancer in a subject, comprising
administering to the subject an effective amount of the antigen
binding protein of claim 28, optionally wherein the cancer is
selected from a solid tumor and a hematological tumor
Description
CROSS REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/719,565, filed Aug. 17, 2018, U.S. Provisional
Application No. 62/808,775, filed Feb. 21, 2019, and U.S.
Provisional Application No. 62/869,923, filed Jul. 2, 2019, which
applications are hereby incorporated by reference in their
entirety.
RELATED APPLICATIONS
[0002] This application is related to PCT/US2018/046997, filed on
Aug. 17, 2018, and to PCT/US2018/06793, filed on Dec. 28, 2018,
which applications are incorporated by reference in their
entirety.
SEQUENCE LISTING
[0003] [insert]
BACKGROUND
[0004] The immune system employs two types of adaptive immune
responses to provide antigen specific protection from pathogens;
humoral immune responses, and cellular immune responses, which
involve specific recognition of pathogen antigens via B lymphocytes
and T lymphocytes, respectively.
[0005] T lymphocytes, by virtue of being the antigen specific
effectors of cellular immunity, play a central role in the body's
defense against diseases mediated by intracellular pathogens, such
as viruses, intracellular bacteria, mycoplasmas, and intracellular
parasites, and against cancer cells by directly cytolysing the
affected cells. The specificity of T lymphocyte responses is
conferred by, and activated through T-cell receptors (TCRs) binding
to (major histocompatibility complex) MHC molecules on the surface
of affected cells. T-cell receptors are antigen specific receptors
clonally distributed on individual T lymphocytes whose repertoire
of antigenic specificity is generated via somatic gene
rearrangement mechanisms analogous to those involved in generating
the antibody gene repertoire. T-cell receptors include a
heterodimer of transmembrane molecules, the main type being
composed of an alpha-beta polypeptide dimer and a smaller subset of
a gamma-delta polypeptide dimer. T lymphocyte receptor subunits
comprise a variable and constant region similar to immunoglobulins
in the extracellular domain, a short hinge region with cysteine
that promotes alpha and beta chain pairing, a transmembrane and a
short cytoplasmic region. Signal transduction triggered by TCRs is
indirectly mediated via CD3-zeta, an associated multi-subunit
complex comprising signal transducing subunits.
[0006] T lymphocyte receptors do not generally recognize native
antigens but rather recognize cell-surface displayed complexes
comprising an intracellularly processed fragment of an antigen in
association with a major histocompatibility complex (MHC) for
presentation of peptide antigens. Major histocompatibility complex
genes are highly polymorphic across species populations, comprising
multiple common alleles for each individual gene. In humans, MHC is
referred to as human leukocyte antigen (HLA).
[0007] Major histocompatibility complex class I molecules are
expressed on the surface of virtually all nucleated cells in the
body and are dimeric molecules comprising a transmembrane heavy
chain, comprising the peptide antigen binding cleft, and a smaller
extracellular chain termed beta2-microglobulin. MHC class I
molecules present peptides derived from the degradation of
cytosolic proteins by the proteasome, a multi-unit structure in the
cytoplasm, (Niedermann G., 2002. Curr Top Microbiol Immunol.
268:91-136; for processing of bacterial antigens, refer to Wick M
J, and Ljunggren H G., 1999. Immunol Rev. 172:153-62). Cleaved
peptides are transported into the lumen of the endoplasmic
reticulum (ER) by the transporter associated with antigen
processing (TAP) where they are bound to the groove of the
assembled class I molecule, and the resultant MHC/peptide complex
is transported to the cell membrane to enable antigen presentation
to T lymphocytes (Yewdell J W., 2001. Trends Cell Biol. 11:294-7;
Yewdell J W. and Bennink J R., 2001. Curr Opin Immunol. 13:13-8).
Alternatively, cleaved peptides can be loaded onto MHC class I
molecules in a TAP-independent manner and can also present
extracellularly-derived proteins through a process of
cross-presentation. As such, a given MHC/peptide complex presents a
novel protein structure on the cell surface that can be targeted by
a novel antigen-binding protein (e.g., antibodies or TCRs) once the
identity of the complex's structure (peptide sequence and MHC
subtype) is determined.
[0008] Tumor cells can express antigens and may display such
antigens on the surface of the tumor cell. Such tumor-associated
antigens can be used for development of novel immunotherapeutic
reagents for the specific targeting of tumor cells. For example,
tumor-associated antigens can be used to identify therapeutic
antigen binding proteins, e.g., TCRs, antibodies, or
antigen-binding fragments. Such tumor-associated antigens may also
be utilized in pharmaceutical compositions, e.g., vaccines.
SUMMARY
[0009] Provided herein is an isolated antigen binding protein (ABP)
that specifically binds to a human leukocyte antigen (HLA)-PEPTIDE
target, wherein the HLA-PEPTIDE target comprises an HLA-restricted
peptide complexed with an HLA Class I molecule, wherein the
HLA-restricted peptide is located in the peptide binding groove of
an .alpha.1/.alpha.2 heterodimer portion of the HLA Class I
molecule, wherein the HLA Class I molecule is HLA subtype B*35:01
(reference sequence:
MGSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRTEPRAPWIEQE
GPEYWDRNTQIFKTNTQTYRESLRNLRGYYNQSEAGSHIIQRMYGCDLGPDGRLLR
GHDQSAYDGKDYIALNEDLSSWTAADTAAQITQRKWEAARVAEQLRAYLEGLCVE
WLRRYLENGKETLQRADPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGE
DQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLR) and the
HLA-restricted peptide comprises the sequence EVDPIGHVY, and
wherein the ABP binds to any one or more of: (a) any one or more of
amino acid positions 2-9 of the restricted peptide EVDPIGHVY; (b)
any one or more of amino acid positions 50, 54, 55, 57, 61, 62, 74,
81, 82 and 85 of the .alpha.1 helix of HLA subtype B*35:01; and (c)
any one or more of amino acid positions 147 and 148 of the .alpha.2
helix of HLA subtype B*35:01. Note that recited ranges include
terminal residues. For example, an ABP that binds to any one or
more of positions 2-9 of the restricted peptide EVDPIGHVY contacts
at least one of residues 2, 3, 4, 5, 6, 7, 8, and 9 of the
restricted peptide EVDPIGHVY.
[0010] In some embodiments, the ABP binds to any one or more of
amino acid positions 2-8 of the restricted peptide EVDPIGHVY
[0011] In some embodiments, the ABP binds to any one or more of
amino acid positions 5-9 of the restricted peptide EVDPIGHVY.
[0012] In some embodiments, the HLA Class I molecule is HLA subtype
B*35:01 and the HLA-restricted peptide consists of the sequence
EVDPIGHVY
[0013] In some embodiments, the ABP comprises a CDR-H3 comprising a
sequence selected from: CARDGVRYYGMDVW, CARGVRGYDRSAGYW,
CASHDYGDYGEYFQHW, CARVSWYCSSTSCGVNWFDPW, CAKVNWNDGPYFDYW,
CATPTNSGYYGPYYYYGMDVW, CARDVMDVW, CAREGYGMDVW, CARDNGVGVDYW,
CARGIADSGSYYGNGRDYYYGMDVW, CARGDYYFDYW, CARDGTRYYGMDVW,
CARDVVANFDYW, CARGHSSGWYYYYGMDVW, CAKDLGSYGGYYW,
CARSWFGGFNYHYYGMDVW, CARELPIGYGMDVW, and CARGGSYYYYGMDVW.
[0014] In some embodiments, the ABP comprises a CDR-L3 comprising a
sequence selected from: CMQGLQTPITF, CMQALQTPPTF, CQQAISFPLTF,
CQQANSFPLTF, CQQANSFPLTF, CQQSYSIPLTF, CQQTYMMPYTF, CQQSYITPWTF,
CQQSYITPYTF, CQQYYTTPYTF, CQQSYSTPLTF, CMQALQTPLTF, CQQYGSWPRTF,
CQQSYSTPVTF, CMQALQTPYTF, CQQANSFPFTF, CMQALQTPLTF, and
CQQSYSTPLTF.
[0015] In some embodiments, the ABP comprises the CDR-H3 and the
CDR-L3 from the scFv designated G5_P7_E7, G5_P7_B3, G5_P7_A5,
G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08,
G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8,
G5R4-P3G05, G5R4-P4A07, or G5R4-P4B01.
[0016] In some embodiments, the ABP comprises all three heavy chain
CDRs and all three light chain CDRs from the scFv designated
G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12,
G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06,
G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, or
G5R4-P4B01.
[0017] In some embodiments, the ABP comprises a VH sequence
selected from
TABLE-US-00001 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGI
INPRSGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDG
VRYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSHDINWVRQAPGQGLEWMGW
MNPNSGDTGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGV
RGYDRSAGYWGQGTLVIVSS,
EVQLLESGGGLVKPGGSLRLSCAASGFSFSSYWMSWVRQAPGKGLEWISY
ISGDSGYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCASHD
YGDYGEYFQHWGQGTLVTVSS,
EVQLLQSGGGLVQPGGSLRLSCAASGFTFSNSDMNWVRQAPGKGLEWVAY
ISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVS
WYCSSTSCGVNWFDPWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNSDMNWVRQAPGKGLEWVAS
ISSSGGYINYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVN
WNDGPYFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNFGVSWLRQAPGQGLEWMGG
IIPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATPT
NSGYYGPYYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGW
INPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDV MDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSGYLVSWVRQAPGQGLEWMGW
INPNSGGTNTAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREG
YGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYIFRNYPMHWVRQAPGQGLEWMGW
INPDSGGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDN
GVGVDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGW
MNPNIGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGI
ADSGSYYGNGRDYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGW
INPNSGVTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGD
YYFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGW
INPNSGDTKYSQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDG
TRYYGMDVWGQGTTVTVSS,
EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSY
ISSSSSYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDV
VANFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGW
MNPDSGSTGYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGH
SSGWYYYYGMDVWGQGTTVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFTSYSMHWVRQAPGKGLEWVSS
ITSFTNTMYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDL
GSYGGYYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGI
INPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSW
FGGFNYHYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGW
MNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREL
PIGYGMDVWGQGTTVTVSS, and
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IIPIVGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
SYYYYGMDVWGQGTTVTVSS.
[0018] In some embodiments, the ABP comprises a VL sequence
selected from
TABLE-US-00002 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTP ITFGQGTRLEIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSSRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP PTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAISFPLTFGQ STKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYS
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYY
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYMMPYTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYGAS
SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPWTFGQGT KVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPYTFGQ GTKLEIK,
DIVMTQSPDSLAVSLGERATINCKTSQSVLYRPNNENYLAWYQQKPGQPP
KLLIYQASIREPGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTT PYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRFLNWYQQKPGKAPKLLIYG
ASRPQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQ GTKVEIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSHRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP LTFGGGTKVEIK,
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYA
ASARASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSWPRTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYGAS
RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPVTFGQGT KVEIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP YTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASEDISNHLNWYQQKPGKAPKWYDAL
SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGT KVDIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP LTFGQGTKVEIK,
and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKVEIK.
[0019] In some embodiments, the ABP comprises the VH sequence and
VL sequence from the scFv designated G5_P7_E7, G5_P7_B3, G5_P7_A5,
G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08,
G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8,
G5R4-P3G05, G5R4-P4A07, and G5R4-P4B01.
[0020] Also provided herein is an isolated antigen binding protein
(ABP) that specifically binds to a human leukocyte antigen
(HLA)-PEPTIDE target, wherein the HLA-PEPTIDE target comprises an
HLA-restricted peptide complexed with an HLA Class I molecule,
wherein the HLA-restricted peptide is located in the peptide
binding groove of an .alpha.1/.alpha.2 heterodimer portion of the
HLA Class I molecule, the HLA Class I molecule is HLA subtype
A*01:01 (reference sequence: MGSHSMRYFF
TSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQKMEPRAPWIEQEG
PEYWDQETRNMKAHSQTDRANLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLR
GYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAVHAAEQRRVYLEGRCV
DGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGE
DQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLR), and the
HLA-restricted peptide comprises the sequence NTDNNLAVY, and
wherein the ABP binds to any one or more of: (a) any one or more of
residues 3-9 of the restricted peptide NTDNNLAVY, (b) any one or
more of residues 70-85 of the of the alpha 1 helix of HLA subtype
allele A*01:01, and (c) any one or more of residues 140-160 of the
alpha 2 helix of HLA subtype allele A*01:01.
[0021] In some embodiments, the ABP binds to any one or more of
residues 6-9 of the restricted peptide NTDNNLAVY
[0022] In some embodiments, the ABP binds to any one or more of
residues 7-8 of the restricted peptide NTDNNLAVY
[0023] In some embodiments, the ABP binds to one or more of
residues 157-160 of the alpha 2 helix of HLA subtype allele
A*01:01.
[0024] In some embodiments, the ABP binds to one or more of
residues 6-9 of the restricted peptide NTDNNLAVY and one or more of
residues 157-160 of the alpha 2 helix of the HLA subtype allele
A*01:01.
[0025] In some embodiments, the HLA Class I molecule is HLA subtype
A*01:01 and the HLA-restricted peptide consists of the sequence
NTDNNLAVY
[0026] In some embodiments, the ABP comprises a CDR-H3 comprising a
sequence selected from: CAATEWLGVW, CARANWLDYW, CARANWLDYW,
CARDWVLDYW, CARGEWLDYW, CARGWELGYW, CARDFVGYDDW, CARDYGDLDYW,
CARGSYGMDVW, CARDGYSGLDVW, CARDSGVGMDVW, CARDGVAVASDYW,
CARGVNVDDFDYW, CARGDYTGNWYFDLW, CARANWLDYW, CARDQFYGGNSGGHDYW,
CAREEDYW, CARGDWFDPW, CARGDWFDPW, CARGEWFDPW, CARSDWFDPW,
CARDSGSYFDYW, CARDYGGYVDYW, CAREGPAALDVW, CARERRSGMDVW,
CARVLQEGMDVW, CASERELPFDIW, CAKGGGGYGMDVW, CAAMGIAVAGGMDVW,
CARNWNLDYW, CATYDDGMDVW, CARGGGGALDYW, CALSGNYYGMDVW,
CARGNPWELRLDYW, and CARDKNYYGMDVW.
[0027] In some embodiments, the ABP comprises a CDR-L3 comprising a
sequence selected from: CQQSYNTPYTF, CQQSYSTPYTF, CQQSYSTPYSF,
CQQSYSTPFTF, CQQSYGVPYTF, CQQSYSAPYTF, CQQSYSAPYTF, CQQSYSAPYSF,
CQQSYSTPYTF, CQQSYSVPYSF, CQQSYSAPYTF, CQQSYSVPYSF, CQQSYSTPQTF,
CQQLDSYPFTF, CQQSYSSPYTF, CQQSYSTPLTF, CQQSYSTPYSF, CQQSYSTPYTF,
CQQSYSTPYTF, CQQSYSTPFTF, CQQSYSTPTF, CQQTYAIPLTF, CQQSYSTPYTF,
CQQSYIAPFTF, CQQSYSIPLTF, CQQSYSNPTF, CQQSYSTPYSF, CQQSYSDQWTF,
CQQSYLPPYSF, CQQSYSSPYTF, CQQSYTTPWTF, CQQSYLPPYSF, CQEGITYTF,
CQQYYSYPFTF, and CQHYGYSPVTF.
[0028] In some embodiments, the ABP comprises the CDR-H3 and the
CDR-L3 from the scFv designated G2-P1H11, G2-P2E07, G2-P2E03,
G2-P2A11, G2-P2C06, G2-P1G01, G2-P1C02, G2-P1H01, G2-P1B12,
G2-P1B06, G2-P2H10, G2-P1H10, G2-P2C11, G2-P1C09, G2-P1A10,
G2-P1B10, G2-P1D07, G2-P1E05, G2-P1D03, G2-P1G12, G2-P2H11,
G2-P1C03, G2-P1G07, G2-P1F12, G2-P1G03, G2-P2B08, G2-P2A10,
G2-P2D04, G2-P1C06, G2-P2A09, G2-P1B08, G2-P1E03, G2-P2A03,
G2-P2F01, or G2-P1D06.
[0029] In some embodiments, the ABP comprises the CDR-H3 and the
CDR-L3 from the scFv designated G2-P1H11.
[0030] In some embodiments, the ABP comprises all three heavy chain
CDRs and all three light chain CDRs from the scFv designated
G2-P1H11, G2-P2E07, G2-P2E03, G2-P2A11, G2-P2C06, G2-P1G01,
G2-P1C02, G2-P1H01, G2-P1B12, G2-P1B06, G2-P2H10, G2-P1H10,
G2-P2C11, G2-P1C09, G2-P1A10, G2-P1B10, G2-P1D07, G2-P1E05,
G2-P1D03, G2-P1G12, G2-P2H11, G2-P1C03, G2-P1G07, G2-P1F12,
G2-P1G03, G2-P2B08, G2-P2A10, G2-P2D04, G2-P1C06, G2-P2A09,
G2-P1B08, G2-P1E03, G2-P2A03, G2-P2F01, or G2-P1D06.
[0031] In some embodiments, the ABP comprises all three heavy chain
CDRs and all three light chain CDRs from the scFv designated
G2-P1H11.
[0032] In some embodiments, the ABP comprises a VH sequence
selected from
TABLE-US-00003 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMEIWVRQAPGQGLEWM
GMINPSGGGTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCA
RGNPWELRLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSATISWVRQAPGQGLEWMG
WIYPNSGGTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAA
TEWLGVWGQGTTVTVSS,
EVQLLQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG
WINPNSGGTISAPNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
ANWLDYWGQGTLVTVSS,
EVQLLESGAEVKKPGASVKVSCKASGYTFTTYDLAWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
ANWLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKSSGYSFDSYVVNWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DWVLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMG
WMNPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GEWLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GWELGYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTINWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DFVGYDDWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGITWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DYGDLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSNYILSWVRQAPGQGLEWMG
WINPDSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GSYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYSFTRYNMHWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DGYSGLDVWGKGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG
WINPNNGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DSGVGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFNNYAFSWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DGVAVASDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSSYNMHWVRQAPGQGLEWMG
WINGNTGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GVNVDDFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMG
WINPDTGYTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GDYTGNWYFDLWGRGTLVTVSS,
EVQLLESGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMG
WINPYSGGTNYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
ANWLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMG
WISAYNGYTNYAQNLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DQFYGGNSGGHDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQGLEWMG
WMNPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR E-EDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTINWVRQAPGQGLEWMG
WINPNSGGANYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GDWFDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLMHWVRQAPGQGLEWMG
WISPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GDWFDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYVHWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GEWFDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMHWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
SDWFDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSNYAINWVRQAPGQGLEWMG
WISPYSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DSGSYFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMG
WIYPNTGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DYGGYVDYWGQGTLVTVSS,
EVQLLESGAEVKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMG
WMNPNSGGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
EGPAALDVWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTLTSHLIHWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
ERRSGMDVWGQGTTVTVSS,
EVQLLESGAEVKKPGASVKVSCKASGYSFTDYIVHWVRQAPGQGLEWMG
WINPYSGGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
VLQEGMDVWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSNFLINWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAS
ERELPFDIWGQGTMVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYQMFWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAK
GGGGYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAA
MGIAVAGGMDVWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYHMHWVRQAPGQGLEWMG
WIHPDSGGTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
NWNLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMG
WMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAT
YDDGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTVNWVRQAPGQGLEWMG
WINPNSGGTKYAQNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GGGGALDWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG
MINPRDDTTDYARDFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAL
SGNYYGMDVWGQGTTVTVSS, and
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSQYMHWVRQAPGQGLEWMG
RIIPLLGIVNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR
DKNYYGMDVWGQGTTVTVSS.
[0033] In some embodiments, the ABP comprises a VL sequence
selected from
TABLE-US-00004
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSG
TDFTLTISSLQPEDFATYYCQQYYSYPFTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYAASSLRSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYAASTVQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQDISRWLAWYQQKPGKAPKLLIYAASRLQAGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQTISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQTISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYGVPYTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISNWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSVGNWLAWYQQKPGKAPKLLIYGASSLQTGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQNIGNWLAWYQQKPGKAPKLLIYAASTLQTGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYGASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSVPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISKWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSVPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQTISNYLNWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPQTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASRDIGRAVGWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQLDSYPFTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPYTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSIGRWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYSFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFAQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYGASRLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSVSNWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQTYAIPLTFGGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDIGSWLAWYQQKPGKAPKLLIYATSSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIYAASTLQPGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYIAPFTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASRLESGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTFGGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYGVSSLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWVAWYQQKPGKAPKLLIYGASNLESGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSDQWTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYAASSLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYLPPYSFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISNWLAWYQQKPGKAPKLLIYAASSLQSGVPS
RFSGSGSGTYFTLTISSLQPEDFATYYCQQSYSSPYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISHYLNWYQQKPGKAPKLLIYGASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPWTFGQGTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASTLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYLPPYSFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYGASRLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQEGITYTFGQGTKVEIK, and
EIVMTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPGQAPRLLIYGASTRATGIPAR
FSGSGSGTEFTLTISSLQSEDFAVYYCQHYGYSPVTFGQGTKLEIK.
[0034] In some embodiments, the ABP comprises the VH sequence and
the VL sequence from the scFv designated G2-P1H11, G2-P2E07,
G2-P2E03, G2-P2A11, G2-P2C06, G2-P1G01, G2-P1C02, G2-P1H01,
G2-P1B12, G2-P1B06, G2-P2H10, G2-P1H10, G2-P2C11, G2-P1C09,
G2-P1A10, G2-P1B10, G2-P1D07, G2-P1E05, G2-P1D03, G2-P1G12,
G2-P2H11, G2-P1C03, G2-P1G07, G2-P1F12, G2-P1G03, G2-P2B08,
G2-P2A10, G2-P2D04, G2-P1C06, G2-P2A09, G2-P1B08, G2-P1E03,
G2-P2A03, G2-P2F01, or G2-P1D06.
[0035] In some embodiments, the ABP comprises the VH sequence and
the VL sequence from the scFv designated G2-P1H11.
[0036] Also provided herein is an isolated antigen binding protein
(ABP) that specifically binds to a human leukocyte antigen
(HLA)-PEPTIDE target, wherein the HLA-PEPTIDE target comprises an
HLA-restricted peptide complexed with an HLA Class I molecule,
wherein the HLA-restricted peptide is located in the peptide
binding groove of an .alpha.1/.alpha.2 heterodimer portion of the
HLA Class I molecule, wherein the HLA Class I molecule is HLA
subtype A*02:01 (reference sequence:
TABLE-US-00005 MGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVREDSDAASQRMEPRA
PWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMY
GCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEA
AHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEA
TLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVV
PSGQEQRYTCHVQHEGLPKPLTLR),
and the HLA-restricted peptide comprises the sequence AIFPGAVPAA,
and wherein the ABP binds to any one or more of: (a) any one or
more of amino acid positions 1-6 of the restricted peptide
AIFPGAVPAA, (b) any one or more of amino acid positions 46, 49, 55,
61, 74, 76, 77, 78, 81 and 84 of the .alpha.1 helix of HLA subtype
A*02:01, (c) any one or more of amino acid positions 45-60, 66, 67,
and 73 of the .alpha.1 helix of HLA subtype A*02:01, (d) any one or
more of amino acid positions 138, 145, 147, 152-156, 164, 167 of
the .alpha.2 helix of HLA subtype A*02:01, and (e) any one or more
of any one or more of amino acid positions 56, 59, 60, 63, 64, 66,
67, 70, 73, 74, 132, 150-153, 155, 156, 158-160, 162-164, 166-168,
170, and 171 of HLA subtype A*02:01.
[0037] In some embodiments, the ABP binds to any one or more of
amino acid positions 1-5 of the restricted peptide AIFPGAVPAA.
[0038] In some embodiments, the ABP binds to one or both of amino
acid positions 4 and 5 of the restricted peptide AIFPGAVPAA.
[0039] In some embodiments, the ABP binds to one or both of amino
acid positions 5 and 6 of the restricted peptide AIFPGAVPAA.
[0040] In some embodiments, the ABP binds to amino acid position 6
of the restricted peptide AIFPGAVPAA.
[0041] In some embodiments, the ABP binds to any one or more of
amino acid positions 46, 49, 55, 66, 67, and 73 of the .alpha.1
helix of HLA subtype A*02:01.
[0042] In some embodiments, the ABP comprises a VH region
comprising a paratope comprising at least one, two, three, or four
of residues Tyr32, Gly99, Asp100, and Tyr100A of the VH region
shown in the sequence
QVQLVQSGAEVKKPGASVKVSCKASGGTLSSYPINWVRQAPGQGLEWMGWISTYS
GHADYAQKLQGRVTMTRDTSTSTVYMEL SSLRSEDTAVYYCARSYDYGDYLNFDY
WGQGTLVTVSS, as numbered by the Kabat numbering system.
[0043] In some embodiments, the ABP comprises a VH region
comprising a paratope comprising at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 of
residues Thr28, Leu 29, Ser 30, Ser 31, Tyr 32, Pro 33, Trp 47, Trp
50, Ser 52, Tyr 53, Ser 54, His 56, Asp 58, Tyr 59, Gln 61, Gln 64,
Asp 97, Tyr 98, Gly 99, Asp100, Tyr100A, Leu100B, and Asn100C of
the VH region shown in the sequence
QVQLVQSGAEVKKPGASVKVSCKASGGTLSSYPINWVRQAPGQGLEWMGWISTYS
GHADYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSYDYGDYLNFDY
WGQGTLVTVSS, as numbered by the Kabat numbering system.
[0044] In some embodiments, the paratope comprises at least 1, 2,
3, 4, 5, 6, or 7 of residues Ser 30, Ser 31, Tyr 32, Tyr 98, Gly
99, Asp 100, and Tyr 100A of the VH region, as numbered by the
Kabat numbering system.
[0045] In some embodiments, the ABP comprises a VL region
comprising a paratope comprising at least one, two, or three of
residues Tyr32, Ser 91, and Tyr 92 of the VL region shown in the
sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPPTFGGGTKVDIK, as numbered by
the Kabat numbering system.
[0046] In some embodiments, the ABP comprises a VL region
comprising a paratope comprising at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, or 13 of residues Asp1, Ser30, Asn31, Tyr32, Tyr49,
Ala50, Ser53, Ser67, Ser91, Tyr92, Ser93, Ile94, and Pro95 of the
VL region shown in the sequence
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYAASSLQSGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPPTFGGGTKVDIK, as numbered by
the Kabat numbering system.
[0047] In some embodiments, the paratope comprises at least 1, 2,
3, 4, 5, or 6 of residues Asp1, Asn31, Tyr32, Ser91, Tyr92, and
Ile94 of the VL region, as numbered by the Kabat numbering
system.
[0048] In some embodiments, the HLA Class I molecule is HLA subtype
A*02:01 and the HLA-restricted peptide consists of the sequence
AIFPGAVPAA.
[0049] In some embodiments, the ABP comprises a CDR-H3 comprising a
sequence selected from: CARDDYGDYVAYFQHW, CARDLSYYYGMDVW,
CARVYDFWSVLSGFDIW, CARVEQGYDIYYYYYMDVW, CARSYDYGDYLNFDYW,
CARASGSGYYYYYGMDVW, CAASTWIQPFDYW, CASNGNYYGSGSYYNYW,
CARAVYYDFWSGPFDYW, CAKGGIYYGSGSYPSW, CARGLYYMDVW,
CARGLYGDYFLYYGMDVW, CARGLLGFGEFLTYGMDVW, CARDRDSSWTYYYYGMDVW,
CARGLYGDYFLYYGMDVW, CARGDYYDSSGYYFPVYFDYW, and
CAKDPFWSGHYYYYGMDVW.
[0050] In some embodiments, the ABP comprises a CDR-L3 comprising a
sequence selected from: CQQNYNSVTF, CQQSYNTPWTF, CGQSYSTPPTF,
CQQSYSAPYTF, CQQSYSIPPTF, CQQSYSAPYTF, CQQHNSYPPTF, CQQYSTYPITI,
CQQANSFPWTF, CQQSHSTPQTF, CQQSYSTPLTF, CQQSYSTPLTF, CQQTYSTPWTF,
CQQYGSSPYTF, CQQSHSTPLTF, CQQANGFPLTF, and CQQSYSTPLTF.
[0051] In some embodiments, the ABP comprises the CDR-H3 and the
CDR-L3 from the scFv designated G8-P1A03, G8-P1A04, G8-P1A06,
G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06,
R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02,
R3G8-P5G08, G8-P1C01, or G8-P2C11.
[0052] In some embodiments, the ABP comprises all three heavy chain
CDRs and all three light chain CDRs from the scFv designated
G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02,
G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05,
R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11.
[0053] In some embodiments, the ABP comprises a VH sequence
selected from:
TABLE-US-00006
QVQLVQSGAEVKKPGASVKVSCKASGGTFSRSAITWVRQAPGQGLEWMGWINPNSGAT
NYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDDYGDYVAYFQHWGQGT LVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYPFIGQYLHWVRQAPGQGLEWMGIINPSGDSA
TYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDLSYYYGMDVWGQGTTV TVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGWMNPIG
GGTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVYDFWSVLSGFDIWG
QGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSGINWNGGST
GYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVEQGYDIYYYYYMDVWG
KGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTLSSYPINWVRQAPGQGLEWMGWISTYSGH
ADYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSYDYGDYLNFDYWGQG TLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSSISGRGDNT
YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARASGSGYYYYYGMDVWGQ GTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYFMHWVRQAPGQGLEWMGMVNPSGG
SETFAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAASTWIQPFDYWGQGTLVT VSS,
EVQLLESGGGLVQPGGSLRLSCAASGFDFSIYSMNWVRQAPGKGLEWVSAISGSGGSTY
YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASNGNYYGSGSYYNYWGQGTL VTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTLTTYYMHWVRQAPGQGLEWMGWINPNSG
GTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAVYYDFWSGPFDYWGQ
GTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPYSG
GTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKGGIYYGSGSYPSWGQG TLVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGVSWVRQAPGQGLEWMGWISPYSGN
TDYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGLYYMDVWGKGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSNMYLHWVRQAPGQGLEWMGWINPNTG
DTNYAQTFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLYGDYFLYYGMDVW
GQGTKVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWMNPNS
GNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLLGFGEFLTYGMDV
WGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGVINPSGGS
TTYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRDSSWTYYYYGMDVW
GQGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSNYMHWVRQAPGQGLEWMGWMNPNS
GNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLYGDYFLYYGMDV
WGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSHAISWVRQAPGQGLEWMGVIIPSGGTS
YTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYYDSSGYYFPVYFDYWG
QGTLVTVSS, and
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWINPNSG
GTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPFWSGHYYYYGMDV
WGQGTTVTVSS.
[0054] In some embodiments, the ABP comprises a VL sequence
selected from:
TABLE-US-00007 DIQMTQSPSSLSASVGDRVTITCRASQSITSYLNWYQQKPGKAPKWYDAS
NLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYNSVTFGQGTK LEIK,
DIQMTQSPSSLSASVGDRVTITCWASQGISSYLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPWTFGP GTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQAISNSLAWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSTPPTFGQ GTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYK
ASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGP GTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPPTFGG GTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGINSYLAWYQQKPGKAPKWYDAS
NLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNSYPPTFGQGT KLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPITIGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQ GTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQDVSTWLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPQTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYD
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPWTFGQ GTKLEIK,
EIVMTQSPATLSVSPGERATLSCRASQSVGNSLAWYQQKPGQAPRLLIYG
ASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSSPYTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPLTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQNIYTYLNWYQQKPGKAPKLLIYD
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTFGG GTKVEIK, and
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKVEIK.
[0055] In some embodiments, the ABP comprises the VH sequence and
VL sequence from the scFv designated G8-P1A03, G8-P1A04, G8-P1A06,
G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06,
R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02,
R3G8-P5G08, G8-P1C01, or G8-P2C11.
[0056] Also provided herein is an isolated antigen binding protein
(ABP) that specifically binds to a human leukocyte antigen
(HLA)-PEPTIDE target, wherein the HLA-PEPTIDE target comprises an
HLA-restricted peptide complexed with an HLA Class I molecule,
wherein the HLA-restricted peptide is located in the peptide
binding groove of an .alpha.1/.alpha.2 heterodimer portion of the
HLA Class I molecule, wherein the HLA Class I molecule is HLA
subtype A*01:01 (reference sequence: MGSHSMRYFF
TSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQKMEPRAPWIEQEG
PEYWDQETRNMKAHSQTDRANLGTLRGYYNQ SEDGSHTIQIMYGCDVGPDGRFLR
GYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAVHAAEQRRVYLEGRCV
DGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGE
DQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLR), and the
HLA-restricted peptide comprises the sequence ASSLPTTMNY, and
wherein the ABP binds to any one or more of: (a) any one or more of
amino acid positions 4, 6, 7, 8, and 9 of the restricted peptide
ASSLPTTMNY, (b) any one or more of amino acid positions 49-56 of
HLA subtype A*01:01, (c) any one or more of amino acid positions
59-66 of HLA subtype A*01:01, (d) any one or more of amino acid
positions 136-147 of HLA subtype A*01:01, and (e) any one or more
of amino acid positions 157-160 of HLA subtype A*01:01.
[0057] In some embodiments, the ABP binds to any one or more of
amino acid positions 6-9 of the restricted peptide ASSLPTTMNY
[0058] In some embodiments, the ABP binds to any one or more of
amino acid positions 6-7 of the restricted peptide ASSLPTTMNY
[0059] In some embodiments, the ABP binds to amino acid positions 6
of the restricted peptide ASSLPTTMNY.
[0060] In some embodiments, the ABP binds to: (a) any one or more
of amino acid positions 52-54 of HLA subtype A*01:01, (b) any one
or more of amino acid positions 136-139 of HLA subtype A*01:01, (c)
any one or more of amino acid positions 141-147 of HLA subtype
A*01:01, or (d) any one or more of amino acid positions 136-139 and
any one or more of amino acid positions 141-147 of HLA subtype
A*01:01.
[0061] In some embodiments of the ABP comprising an antibody or
antigen-binding fragment thereof, the HLA Class I molecule is HLA
subtype A*01:01 and the HLA-restricted peptide consists of the
sequence ASSLPTTMNY
[0062] In some embodiments, the ABP comprises a CDR-H3 comprising a
sequence selected from: CARDQDTIFGVVITWFDPW, CARDKVYGDGFDPW,
CAREDDSMDVW, CARDSSGLDPW, CARGVGNLDYW, CARDAHQYYDFWSGYYSGTYYYGMDVW,
CAREQWPSYWYFDLW, CARDRGYSYGYFDYW, CARGSGDPNYYYYYGLDVW,
CARDTGDHFDYW, CARAENGMDVW, CARDPGGYMDVW, CARDGDAFDIW, CARDMGDAFDIW,
CAREEDGMDVW, CARDTGDHFDYW, CARGEYSSGFFFVGWFDLW, and
CARETGDDAFDIW.
[0063] In some embodiments, the ABP comprises a CDR-L3 comprising a
sequence selected from: CQQYFTTPYTF, CQQAEAFPYTF, CQQSYSTPITF,
CQQSYIIPYTF, CHQTYSTPLTF, CQQAYSFPWTF, CQQGYSTPLTF, CQQANSFPRTF,
CQQANSLPYTF, CQQSYSTPFTF, CQQSYSTPFTF, CQQSYGVPTF, CQQSYSTPLTF,
CQQSYSTPLTF, CQQYYSYPWTF, CQQSYSTPFTF, CMQTLKTPLSF, and
CQQSYSTPLTF.
[0064] In some embodiments, the ABP comprises the CDR-H3 and the
CDR-L3 from the scFv designated R3G10-P1A07, R3G10-P1B07,
R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04,
R3G10-P2G11, R3G10-P3E04, R3G10-P4A02, R3G10-P4C05, R3G10-P4D04,
R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or
R3G10-P5C08.
[0065] In some embodiments, the ABP comprises all three heavy chain
CDRs and all three light chain CDRs from the scFv designated
R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01,
R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, R3G10-P3E04, R3G10-P4A02,
R3G10-P4C05, R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12,
R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08.
[0066] In some embodiments, the ABP comprises a VH sequence
selected from:
TABLE-US-00008
EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISARSGRT
YYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDQDTIFGVVITWFDPWGQG
TLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIHPGGGT
TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDKVYGDGFDPWGQGTLV TVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYIFTGYYMHWVRQAPGQGLEWMGMIGPSDGS
TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREDDSMDVWGKGTTVTVS S,
QVQLVQSGAEVKKPGASVKVSCKASGYTFIGYYMHWVRQAPGQGLEWMGMIGPSDGS
TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSGLDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPSDG
STSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVGNLDYWGQGTLVTV SS,
QVQLVQSGAEVKKPGASVKVSCKASGVTFSTSAISWVRQAPGQGLEWMGWISPYNGNT
DYAQMLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDAHQYYDFWSGYYSGTY
YYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSNSIINWVRQAPGQGLEWMGWMNPNSGN
TNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREQWPSYWYFDLWGRGTL VTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSTHDINWVRQAPGQGLEWMGVINPSGGS
AIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRGYSYGYFDYWGQGTL VTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGNTFIGYYVHWVRQAPGQGLEWVGIINPNGGSI
SYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSGDPNYYYYYGLDVWG
QGTTVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTLSYYYMHWVRQAPGQGLEWMGMIGPSDG
STSYAQRFQGRVTMTRDTSTGTVYMELSSLRSEDTAVYYCARDTGDHFDYWGQGTLVT VSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGIIGPSDGS
TTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAENGMDVWGQGTTVTV SS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVHWVRQAPGQGLEWMGIIAPSDGS
TNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPGGYMDVWGKGTTVT VSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYLHWVRQAPGQGLEWMGMIGPSDGS
TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGDAFDIWGQGTMVTVS S,
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRISPSDGS
TTYAPKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDMGDAFDIWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGMIGPSDG
STSYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREEDGMDVWGQGTTVTV SS,
QVQLVQSGAEVKKPGASVKVSCKASGYTLSYYYMEIWVRQAPGQGLEWMGMIGPSDG
STSYAQRFQGRVTMTRDTSTGTVYMELSSLRSEDTAVYYCARDTGDHFDYWGQGTLVT VSS,
QVQLVQSGAEVKKPGSSVKVSCKASGGTFNNFAISWVRQAPGQGLEWMGGIIPIFDATN
YAQKFQGRVTFTADESTSTAYMELSSLRSEDTAVYYCARGEYSSGFFFVGWFDLWGRGT QVTVSS,
and QVQLVQSGAEVKKPGASVKVSCKASGYNFTGYYMEIWVRQAPGQGLEWMGIIAPSDGS
TNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARETGDDAFDIWGQGTMVT VSS.
[0067] In some embodiments, the ABP comprises a VL sequence
selected:
TABLE-US-00009 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYA
ASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYFTTPYTFGQ GTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIFD
ASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAEAFPYTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGQ GTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYK
ASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYIIPYTFGQ GTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQTYSTPLTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKWYSAS
NLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPWTFGQGT KVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQNISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSTPLTFGQ GTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQDISRYLAWYQQKPGKAPKLLIYD
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPRTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYA
ASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSLPYTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASTLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGP GTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQRISSYLNWYQQKPGKAPKWYSAS
TLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGT KVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYD
ASKLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGVPTFGQG TKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYD
ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKLLIYD
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSYPWTFGQ GTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASTLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGP GTKVDIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQTLKTP LSFGGGTKVEIK,
and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKVEIK.
[0068] In some embodiments, the ABP comprises the VH sequence and
VL sequence from the scFv designated R3G10-P1A07, R3G10-P1B07,
R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04,
R3G10-P2G11, R3G10-P3E04, R3G10-P4A02, R3G10-P4C05, R3G10-P4D04,
R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or
R3G10-P5C08.
[0069] Also provided herein is an isolated antigen binding protein
(ABP) that specifically binds to a human leukocyte antigen
(HLA)-PEPTIDE target, wherein the HLA-PEPTIDE target comprises an
HLA-restricted peptide complexed with an HLA Class I molecule,
wherein the HLA-restricted peptide is located in the peptide
binding groove of an .alpha.1/.alpha.2 heterodimer portion of the
HLA Class I molecule, wherein the HLA Class I molecule is HLA
subtype A*02:01 (reference sequence:
TABLE-US-00010 MGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVREDSDAASQRMEPRA
PWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMY
GCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEA
AHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEA
TLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVV
PSGQEQRYTCHVQHEGLPKPLTLR),
and the HLA-restricted peptide comprises the sequence LLASSILCA,
and wherein the ABP binds to any one or more of: (a) any one or
more of residues 1-5 of the restricted peptide LLASSILCA, (b) any
one or more of residues 49-85 of the HLA-A*02:01 alpha 1 helix, and
(c) any one or more of residues 57-67 of the HLA-A*02:01 alpha 1
helix.
[0070] In some embodiments of the ABP comprising an antibody or
antigen-binding fragment thereof, the HLA Class I molecule is HLA
subtype A*02:01 and the HLA-restricted peptide consists of the
sequence LLASSILCA.
[0071] In some embodiments, the ABP comprises a CDR-H3 comprising a
sequence selected from: CARDGYDFWSGYTSDDYW, CASDYGDYR,
CARDLMTTVVTPGDYGMDVW, CARQDGGAFAFDIW, CARELGYYYGMDVW,
CARALIFGVPLLPYGMDVW, CAKDLATVGEPYYYYGMDVW, and
CARLWFGELHYYYYYGMDVW.
[0072] In some embodiments, the ABP comprises a CDR-L3 comprising a
sequence selected from: CHHYGRSHTF, CQQANAFPPTF, CQQYYSIPLTF,
CQQSYSTPPTF, CQQSYSFPYTF, CMQALQTPLTF, CQQGNTFPLTF, and
CMQGSHWPPSF.
[0073] In some embodiments, the ABP comprises the CDR-H3 and the
CDR-L3 from the scFv designated G7R3-P1C6, G7R3-P1G10, 1-G7R3-P1B4,
2-G7R4-P2C2, 3-G7R4-P1A3, 4-G7R4-B5-P2E9, 5-G7R4-B10-P1F8, or B7
(G7R3-P3A9).
[0074] In some embodiments, the ABP comprises all three heavy chain
CDRs and all three light chain CDRs from the scFv designated
G7R3-P1C6, G7R3-P1G10, 1-G7R3-P1B4, 2-G7R4-P2C2, 3-G7R4-P1A3,
4-G7R4-B5-P2E9, 5-G7R4-B10-P1F8, or B7 (G7R3-P3A9).
[0075] In some embodiments, the ABP comprises a VH sequence
selected from
TABLE-US-00011 QVQLVQSGAEVKKPGASVKVSCKASGGTFSNYGISWVRQAPGQGLEWMGI
INPGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGY
DFWSGYTSDDYWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSG
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASDY GDYRGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHWVRQAPGQGLEWMGW
LNPNSGNTGYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDL
MTTVVTPGDYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASMKVSCKASGYTFTTDGISWVRQAPGQGLEWMGR
IYPHSGYTEYAKKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARQD
GGAFAFDIWGQGTMVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSQYMHWVRQAPGQGLEWMGW
ISPNNGDTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREL
GYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASRYTFTSYDINWVRQAPGQGLEWMGR
IIPMLNIANYAPKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARAL
IFGVPLLPYGMDVWGQGTTVTVSS,
EVQLLQSGGGLVQPGGSLRLSCAASGFTFSSSWMHWVRQAPGKGLEWVSF
ISTSSGYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDL
ATVGEPYYYYGMDVWGQGTTVTVSS, and
QVQLVQSGAEVKKPGSSVKVSCKASGDTFNTYALSWVRQAPGQGLEWMGW
MNPNSGNAGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLW
FGELHYYYYYGMDVWGQGTMVTVSS.
[0076] In some embodiments, the ABP comprises a VL sequence
selected from
TABLE-US-00012 EIVMTQSPATLSVSPGERATLSCRASQSVSSSNLAWYQQKPGQAPRLLIY
GASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCHHYGRSHTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANAFPPTFGQ GTKVEIK,
DIVMTQSPDSLAVSLGERATINCKSSQSVFYSSNNKNQLAWYQQKPGQPP
KWYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSIPL TFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDIFKYLNWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQ GTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYY
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPYTFGQ GTKVEIK,
DIVMTQSPLSLPVTPGEPASISCSSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP LTFGGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYS
ASNLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTFPLTFGQ GTKVEIK, and
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSHWP
PSFGQGTRLEIK.
[0077] In some embodiments, the ABP comprises the VH sequence and
the VL sequence from the scFv designated G7R3-P1C6, G7R3-P1G10,
1-G7R3-P1B4, 2-G7R4-P2C2, 3-G7R4-P1A3, 4-G7R4-B5-P2E9,
5-G7R4-B10-P1F8, or B7 (G7R3-P3A9).
[0078] In some embodiments, the ABP comprises an antibody or
antigen-binding fragment thereof. In some embodiments, the antigen
binding protein is linked to a scaffold, optionally the scaffold
comprises serum albumin or Fc, optionally wherein Fc is human Fc
and is an IgG (IgG1, IgG2, IgG3, IgG4), an IgA (IgA1, IgA2), an
IgD, an IgE, or an IgM isotype Fc. In some embodiments, the antigen
binding protein is linked to a scaffold via a linker, optionally
the linker is a peptide linker, optionally the peptide linker is a
hinge region of a human antibody. In some embodiments, the antigen
binding protein comprises an Fv fragment, a Fab fragment, a F(ab')2
fragment, a Fab' fragment, an scFv fragment, an scFv-Fc fragment,
and/or a single-domain antibody or antigen binding fragment
thereof. In some embodiments, the antigen binding protein comprises
an scFv fragment. In some embodiments, the antigen binding protein
comprises one or more antibody complementarity determining regions
(CDRs), optionally six antibody CDRs. In some embodiments, the
antigen binding protein comprises an antibody. In some embodiments,
the antigen binding protein is a monoclonal antibody. In some
embodiments, the antigen binding protein is a humanized, human, or
chimeric antibody. In some embodiments, the antigen binding protein
is multispecific, optionally bispecific. In some embodiments, the
antigen binding protein binds greater than one antigen or greater
than one epitope on a single antigen. In some embodiments, the
antigen binding protein comprises a heavy chain constant region of
a class selected from IgG, IgA, IgD, IgE, and IgM. In some
embodiments, the antigen binding protein comprises a heavy chain
constant region of the class human IgG and a subclass selected from
IgG1, IgG4, IgG2, and IgG3. In some embodiments, the antigen
binding protein comprises one or more modifications that extend
half-life. In some embodiments, the antigen binding protein
comprises a modified Fc, optionally the modified Fc comprises one
or more mutations that extend half-life, optionally the one or more
mutations that extend half-life is YTE.
[0079] In some embodiments of the isolated ABP, the ABP comprises a
T cell receptor (TCR) or an antigen-binding portion thereof. In
some embodiments, the TCR or antigen-binding portion thereof
comprises a TCR variable region. In some embodiments, the TCR or
antigen-binding portion thereof comprises one or more TCR
complementarity determining regions (CDRs).
[0080] In some embodiments, the TCR comprises an alpha chain and a
beta chain. In some embodiments, the TCR comprises a gamma chain
and a delta chain.
[0081] In some embodiments, the antigen binding protein is a
portion of a chimeric antigen receptor (CAR) comprising: an
extracellular portion comprising the antigen binding protein; and
an intracellular signaling domain. In some embodiments, the antigen
binding protein comprises an scFv and the intracellular signaling
domain comprises an immunoreceptor tyrosine-based activation motif
(ITAM). In some embodiments, the intracellular signaling domain
comprises a signaling domain of a zeta chain of a CD3-zeta (CD3)
chain.
[0082] In some embodiments, the ABP further comprises a
transmembrane domain linking the extracellular domain and the
intracellular signaling domain. In some embodiments, the
transmembrane domain comprises a transmembrane portion of CD28.
[0083] In some embodiments, the ABP further comprises an
intracellular signaling domain of a T cell costimulatory molecule.
In some embodiments, the T cell costimulatory molecule is CD28,
4-1BB, OX-40, ICOS, or any combination thereof.
[0084] Also provided herein is an isolated polynucleotide encoding
an isolated ABP as described herein.
[0085] In some embodiments of the ABP, the antigen binding protein
binds to the HLA-PEPTIDE target through a contact point with the
HLA Class I molecule and through a contact point with the
HLA-restricted peptide of the HLA-PEPTIDE target. In some
embodiments of the ABP, the binding of the ABP to the amino acid
positions on the restricted peptide or HLA subtype, or the contact
points or residues that impact binding, directly or indirectly, of
the HLA-PEPTIDE target with the ABP are determined via positional
scanning, hydrogen-deuterium exchange, or protein
crystallography.
[0086] In some embodiments, the ABP may be for use as a medicament.
In some embodiments, the ABP may be for use in treatment of cancer,
optionally wherein the cancer expresses or is predicted to express
the HLA-PEPTIDE target. In some embodiments, the ABP may be for use
in treatment of cancer, wherein the cancer is selected from a solid
tumor and a hematological tumor.
[0087] Also provided herein is an ABP which is a conservatively
modified variant of the ABP as described herein. Also provided
herein is an antigen binding protein (ABP) that competes for
binding with the antigen binding protein as described herein. Also
provided herein is an antigen binding protein (ABP) that binds the
same HLA-PEPTIDE epitope bound by the antigen binding protein as
described herein.
[0088] Also provided herein is an engineered cell expressing a
receptor comprising the antigen binding protein as described
herein. In some embodiments, the engineered cell is a T cell,
optionally a cytotoxic T cell (CTL). In some embodiments of the
engineered cell, the antigen binding protein is expressed from a
heterologous promoter.
[0089] Also provided herein is an isolated polynucleotide or set of
polynucleotides encoding the antigen binding protein described
herein or an antigen-binding portion thereof.
[0090] Also provided herein is an isolated polynucleotide or set of
polynucleotides encoding the HLA/peptide targets described
herein.
[0091] Also provided herein is a vector or set of vectors
comprising the polynucleotide or set of polynucleotides described
herein.
[0092] Also provided herein is a host cell comprising the
polynucleotide or set of polynucleotides a described herein, or the
vector or set of vectors described herein, optionally wherein the
host cell is CHO or HEK293, or optionally wherein the host cell is
a T cell.
[0093] Also provided herein is a method of producing an antigen
binding protein comprising expressing the antigen binding protein
with the host cell described herein and isolating the expressed
antigen binding protein.
[0094] Also provided herein is a pharmaceutical composition
comprising the antigen binding protein as described herein and a
pharmaceutically acceptable excipient.
[0095] Also provided herein is a method of treating cancer in a
subject, comprising administering to the subject an effective
amount of the antigen binding protein as described herein or a
pharmaceutical composition described herein, optionally wherein the
cancer is selected from a solid tumor and a hematological tumor. In
some embodiments, the cancer expresses or is predicted to express
the HLA-PEPTIDE target.
[0096] Also provided herein is a kit comprising the antigen binding
protein described herein or a pharmaceutical composition described
herein and instructions for use.
[0097] Also provided herein is a composition comprising at least
one HLA-PEPTIDE target described herein and an adjuvant.
[0098] Also provided herein is a composition comprising at least
one HLA-PEPTIDE target described herein and a pharmaceutically
acceptable excipient.
[0099] Also provided herein is a composition comprising an amino
acid sequence comprising a polypeptide of at least one HLA-PEPTIDE
target disclosed in Table A, Table A1, or Table A2, optionally the
amino acid sequence consisting essentially of or consisting of the
polypeptide.
[0100] Also provided herein is a virus comprising the isolated
polynucleotide or set of polynucleotides as described herein. In
some embodiments, the virus is a filamentous phage.
[0101] Also provided herein is a yeast cell comprising the isolated
polynucleotide or set of polynucleotides as described herein.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0102] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, and accompanying drawings, where:
[0103] FIG. 1 shows the general structure of a Human Leukocyte
Antigen (HLA) Class I molecule. By User atropos235 on
en.wikipedia--Own work, CC BY 2.5,
https://commons.wikimedia.org/w/index.php?curid=1805424
[0104] FIG. 2 depicts exemplary construct elements for cloning TCRs
into expression systems for therapy development.
[0105] FIG. 3 shows the target and minipool negative control design
for HLA-PEPTIDE target "G5".
[0106] FIG. 4 shows the target and minipool negative control design
for HLA-PEPTIDE targets "G8" and "G10".
[0107] FIGS. 5A and 5B show HLA stability results for the G5
counterscreen "minipool" and G5 target.
[0108] FIGS. 6A-6E show HLA stability results for the G5 "complete"
pool counterscreen peptides.
[0109] FIGS. 7A and 7B show HLA stability results for counterscreen
peptides and G8 target.
[0110] FIGS. 8A and 8B show HLA stability results for the G10
counterscreen "minipool" and G10 target.
[0111] FIGS. 9A-9D show HLA stability results for the additional G8
and G10 "complete" pool counterscreen peptides.
[0112] FIGS. 10A-10C show phage supernatant ELISA results,
indicating progressive enrichment of G5-, G8 and G10 binding phage
with successive panning rounds.
[0113] FIG. 11 shows a flow chart describing the antibody selection
process, including criteria and intended application for the scFv,
Fab, and IgG formats.
[0114] FIGS. 12A, 12B, and 12C depict bio-layer interferometry
(BLI) results for Fab clone G5-P7A05 to HLA-PEPTIDE target
B*35:01-EVDPIGHVY, Fab clones R3G8-P2C10 and G8-P1C11 to
HLA-PEPTIDE target A*02:01-AIFPGAVPAA, and Fab clone R3G10-P1B07 to
HLA-PEPTIDE target A*01:01-ASSLPTTMNY.
[0115] FIG. 13 shows a general experimental design for the
positional scanning experiments.
[0116] FIG. 14A shows stability results for the G5 positional
variant-HLAs.
[0117] FIG. 14B shows binding affinity of Fab clone G5-P7A05 to the
G5 positional variant-HLAs.
[0118] FIG. 15A shows stability results for the G8 positional
variant-HLAs.
[0119] FIG. 15B shows binding affinity of Fab clone G8-P2C10 to the
G8 positional variant-HLAs.
[0120] FIG. 16A shows stability results for the G10 positional
variant-HLAs.
[0121] FIG. 16B shows binding affinity of Fab clone G10-P1B07 to
the G10 positional variant-HLAs.
[0122] FIGS. 17A, 17B, and 17C show representative examples of
antibody binding to either G5-, G8- or G10-presenting K562 cells,
as detected by flow cytometry.
[0123] FIGS. 18A-18C show histogram plots of K562 cell binding to
generated target-specific antibodies.
[0124] FIGS. 19A-19C show histogram plots of cell binding assays
using tumor cell lines which express HLA subtypes and target genes
of selected HLA-PEPTIDE targets.
[0125] FIGS. 20A and 20B shows number of target-specific T cells
(A) and number of target-specific unique TCR clonotypes (B) from
tested donors.
[0126] FIG. 21A shows an exemplary heatmap for scFv G8-P1H08,
visualized across the HLA portion of HLA-PEPTIDE target G8 in its
entirety using a consolidated perturbation view. FIG. 21B shows an
example of HDX data from scFv G8-P1H08 plotted on a crystal
structure 1jf1.pdb, available at
http://www.rcsb.org/structure/1JF1.
[0127] FIG. 22A shows heat maps across the HLA .alpha.1 helix for
all ABPs tested for HLA-PEPTIDE target G8 (HLA-A*02:01_AIFPGAVPAA).
FIG. 22B shows heat maps across the HLA .alpha.2 helix for all ABPs
tested for HLA-PEPTIDE target G8 (HLA-A*02:01_AIFPGAVPAA. FIG. 22C
shows resulting heat maps across the restricted peptide AIFPGAVPAA
for all ABPs tested.
[0128] FIG. 23A shows an exemplary heatmap for scFv R3G10-P2G11,
visualized across the HLA portion of HLA-PEPTIDE target G10 in its
entirety using a consolidated perturbation view.
[0129] FIG. 23B shows an example of HDX data from scFv R3G10-P2G11
plotted on a crystal structure PDB5bs0.
[0130] FIG. 23C shows an example of HDX data from scFv G10-P5A08
plotted on a crystal structure PDB5bs0.
[0131] FIG. 24A shows resulting heat maps across the HLA .alpha.1
helix for all ABPs tested for HLA-PEPTIDE target G10
(HLA-A*01:01_ASSLPTTMNY). FIG. 24B shows resulting heat maps across
the HLA .alpha.2 helix for all ABPs tested for HLA-PEPTIDE target
G10 (HLA-A*01:01_ASSLPTTMNY). FIG. 24C shows resulting heat maps
across the restricted peptide ASSLPTTMNY for all ABPs tested.
[0132] FIG. 25 depicts exemplary spectral data for peptide
EVDPIGHVY. The figure contains the peptide fragmentation
information as well as information related to the patient sample,
including HLA types.
[0133] FIG. 26 depicts exemplary spectral data for peptide
AIFPGAVPAA. The figure contains the peptide fragmentation
information as well as information related to the patient sample,
including HLA types.
[0134] FIG. 27 depicts exemplary spectral data for peptide
ASSLPTTMNY. The figure contains the peptide fragmentation
information as well as information related to the patient sample,
including HLA types.
[0135] FIGS. 28A and 28B depict size exclusion chromatography
fractions (A) and SDS-PAGE analysis of the chromatography fractions
under reducing conditions (B).
[0136] FIG. 29 depicts photomicrographs of an exemplary crystal of
a complex comprising Fab clone G8-P1C11 and HLA-PEPTIDE target
A*02:01_AIFPGAVPAA ("G8").
[0137] FIG. 30 depicts the overall structure of a complex formed by
binding of Fab clone G8-P1C11 to HLA-PEPTIDE target
A*02:01_AIFPGAVPAA ("G8").
[0138] FIG. 31 depicts a refinement electron density region of the
crystal structure of Fab clone G8-P1C11 complexed with HLA-PEPTIDE
target A*02:01_AIFPGAVPAA ("G8"), the region depicted corresponding
to the restricted peptide AIFPGAVPAA.
[0139] FIG. 32 depicts a LigPlot of the interactions between the
HLA and restricted peptide. The crystal structure corresponds to
Fab clone G8-P1C11 complexed with HLA-PEPTIDE target
A*02:01_AIFPGAVPAA ("G8").
[0140] FIG. 33 depicts a plot of interacting residues between the
Fab VH and VL chains and the restricted peptide. The crystal
structure corresponds to Fab clone G8-P1C11 complexed with
HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8").
[0141] FIG. 34 depicts a LigPlot of the interactions between the
restricted peptide and Fab chains. The crystal structure
corresponds to Fab clone G8-P1C11 complexed with HLA-PEPTIDE target
A*02:01_AIFPGAVPAA ("G8").
[0142] FIG. 35 depicts a LigPlot of the interactions between the
Fab VH chain and the HLA. The crystal structure corresponds to Fab
clone G8-P1C11 complexed with HLA-PEPTIDE target A*02:01_AIFPGAVPAA
("G8").
[0143] FIG. 36 depicts a LigPlot of the interactions between the
Fab VL chain and the HLA. The crystal structure corresponds to Fab
clone G8-P1C11 complexed with HLA-PEPTIDE target A*02:01_AIFPGAVPAA
("G8").
[0144] FIG. 37 depicts the interface summary of a Pisa analysis of
interactions between HLA and restricted peptide. The crystal
structure corresponds to Fab clone G8-P1C11 complexed with
HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8").
[0145] FIG. 38 depicts Pisa analysis of the interacting residues
between the HLA and restricted peptide. The crystal structure
corresponds to Fab clone G8-P1C11 complexed with HLA-PEPTIDE target
A*02:01_AIFPGAVPAA ("G8").
[0146] FIG. 39 depicts Pisa analysis of the interacting residues
between the Fab VH chain and the restricted peptide. The crystal
structure corresponds to Fab clone G8-P1C11 complexed with
HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8").
[0147] FIG. 40 depicts Pisa analysis of the interacting residues
between the Fab VL chain and the restricted peptide. The crystal
structure corresponds to Fab clone G8-P1C11 complexed with
HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8").
[0148] FIG. 41 depicts the interface summary of a Pisa analysis of
interactions between the Fab VH chain and HLA. The crystal
structure corresponds to Fab clone G8-P1C11 complexed with
HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8").
[0149] FIG. 42 depicts Pisa analysis of the interacting residues
between the Fab VH chain and HLA. The crystal structure corresponds
to Fab clone G8-P1C11 complexed with HLA-PEPTIDE target
A*02:01_AIFPGAVPAA ("G8").
[0150] FIG. 43 depicts the interface summary of a Pisa analysis of
interactions between the Fab VL chain and HLA. The crystal
structure corresponds to Fab clone G8-P1C11 complexed with
HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8").
[0151] FIG. 44 depicts Pisa analysis of the interacting residues
between the Fab VL chain and HLA. The crystal structure corresponds
to Fab clone G8-P1C11 complexed with HLA-PEPTIDE target
A*02:01_AIFPGAVPAA ("G8").
[0152] FIG. 45A depicts an exemplary heatmap of the HLA portion of
the G8 HLA-PEPTIDE complex when incubated with scFv clone G8-P1C11,
visualized in its entirety using a consolidated perturbation
view.
[0153] FIG. 45B depicts an example of the HDX data from scFv
G8-P1C11 plotted on a crystal structure of Fab clone G8-P1C11
complexed with HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8").
[0154] FIG. 46 depicts binding affinity of Fab clone G8-P1C11 to
the G8 positional variant-HLAs.
[0155] FIG. 47 shows histogram plots of K562 cell binding to
G8-P1C11, a target-specific antibody to HLA-PEPTIDE target
A*02:01_AIFPGAVPAA ("G8").
[0156] FIG. 48 depicts an exemplary construct backbone sequence for
cloning TCRs into expression systems for therapy development.
[0157] FIG. 49 depicts an exemplary construct sequence for cloning
a TCR specific for A*0201_LLASSILCA into expression systems for
therapy development.
[0158] FIG. 50 depicts an exemplary construct sequence for cloning
a TCR specific for A*0101_EVDPIGHLY into expression systems for
therapy development.
[0159] FIG. 51 shows spectra data for peptide EVDPIGHLY. The figure
contains the peptide fragmentation information as well as
information related to the patient sample, including HLA types.
[0160] FIG. 52 shows spectra data for peptide GVHGGILNK. The figure
contains the peptide fragmentation information as well as
information related to the patient sample, including HLA types.
[0161] FIG. 53 shows spectra data for peptide GVYDGEEHSV.
[0162] FIG. 54 shows spectra data for peptide NTDNNLAVY.
[0163] FIGS. 55-63 show spectra data for additional peptides
disclosed in Table A.
[0164] FIG. 64 shows the design of target screen 1 for the G2
target HLA-A*01:01 NTDNNLAVY.
[0165] FIG. 65A shows the target and minipool negative control
design for the G2 target.
[0166] FIG. 65B shows stability ELISA results for the G2
counterscreen "minipool" and G2 targets.
[0167] FIG. 66 shows stability ELISA results for the additional G2
"complete" pool counterscreen peptides.
[0168] FIG. 67 shows the design of target screen 2 for the G7
target HLA-A*02:01_LLASSILCA.
[0169] FIG. 68 shows stability ELISA results for the additional G7
"complete pool" counterscreen peptides.
[0170] FIG. 69A shows the target and minipool negative control
design for the G7 target.
[0171] FIG. 69B shows stability ELISA results for the G7
counterscreen "minipool" and G7 targets.
[0172] FIGS. 70A and 70B show phage panning results for the G2 and
G7 targets, respectively.
[0173] FIGS. 71A and 71B show biolayer interferometry (BLI) results
for G2 target Fab clone G-2P1H11 and G7 target G7R4-B5-P2E9,
respectively.
[0174] FIG. 72 shows a map of the amino acid substitutions for the
positional scanning experiment described herein.
[0175] FIG. 73A shows a stability heat map for the G2 positional
variant-HLAs.
[0176] FIG. 73B shows an affinity heat map for Fab clone
G2-P1H11.
[0177] FIG. 74A shows a stability heat map for the G7 positional
variants.
[0178] FIG. 74B shows an affinity heat map for Fab clone
G7R4-B5-P2E9.
[0179] FIG. 75 shows cell binding results for Fab clones G2-P1H11
and G7R4-B5-P2E9 to HLA-transduced K562 cells pulsed with target or
negative control peptides.
[0180] FIG. 76 shows cell binding results for Fab clones G2-P1H11
and G7R4-B5-P2E9 to HLA-transduced K562 cells pulsed with target or
negative control peptides.
[0181] FIG. 77 shows an example of hydrogen-deuterium exchange
(HDX) data plotted on a crystal structure PDB 5bs0.
[0182] FIG. 78 shows an exemplary HDX heatmap for scFv clone
G2-P1G07 visualized in its entirety using a consolidated
perturbation view.
[0183] FIG. 79 shows HDX heat maps across the HLA .alpha.1 and
.alpha.2 helices for the tested G2 scFv and Fab clones.
[0184] FIG. 80 shows an HDX heat map across the restricted peptide
NTDNNLAVY for the tested G2 scFv and Fab clones.
[0185] FIG. 81 depicts an experimental workflow by which TCRs which
specifically bind HLA-PEPTIDE targets were isolated.
[0186] FIG. 82 shows a flow cytometry sorting procedure for sorting
MHC-target-specific CD8+ T cells.
[0187] FIG. 83 shows flow cytometry results for exemplary
HLA-PEPTIDE targets B*44:02_GEMSSNSTAL and A*01:01_EVDPIGHLY.
[0188] FIG. 84 shows flow cytometry results for the HLA-PETPIDE
target A*03:01_GVHGGILNK.
[0189] FIG. 85A shows total number of isolated CD8+ T cells per
HLA-PEPTIDE target summed across all donors tested.
[0190] FIG. 85B shows frequency of isolated CD8+ T cells per
HLA-PEPTIDE target summed across all donors tested.
[0191] FIG. 86A depicts the number of unique TCR clonotypes per
HLA-PEPTIDE target for each tested donor.
[0192] FIG. 86B depicts the total number of unique clonotypes per
HLA-PEPTIDE target, summed across all donors tested.
[0193] FIG. 87 shows examples of Jurkat cells expressing
A*0201_LLASSILCA, A*0201_GVYDGEEHSV, B*4402_GEMSSNSTAL, and
A*0101_EVDPIGHLY-specific TCRs binding to their respective
HLA-PEPTIDE targets but not to the control peptide tetramer.
[0194] FIG. 88 shows the gating strategy and flow data
demonstrating that human CD8+ cells transduced with TCRs identified
herein bind to their specific HLA-PEPTIDE target.
[0195] FIG. 89 shows an exemplary lentiviral vector useful for
transducing recipient cells with a TCR disclosed herein.
[0196] FIG. 90 shows BLI results for G2 target Fab clone
G2-P2C06.
[0197] FIG. 91A depicts stability results from a second experiment
for the G2 positional variant-HLAs.
[0198] FIG. 91B depicts binding affinity of Fab clone G2-P2C06 to
the G2 positional variant-HLAs.
[0199] FIG. 92 shows HDX heat maps from a second round of HDX
experiments across the HLA .alpha.1 helix, the HLA .alpha.2 helix,
and the restricted peptide ASSLPTTMNY for various G10 ABPs
tested.
[0200] FIG. 93 shows HDX heat maps from a second round of HDX
experiments across the HLA .alpha.1 helix, the HLA .alpha.2 helix,
and the restricted peptide NTDNNLAVY for G2 ABPs tested.
[0201] FIG. 94 shows an example of HDX data from scFv G2-P2C11
plotted on a crystal structure PDB 5bs0.
[0202] FIG. 95 shows high resolution HDX data plotted on a crystal
structure PDB 5bs0. Data for G2 bound to four different scFvs were
obtained by fragmenting peptides by Electron Transfer Dissociation
(ETD) as described in the Experimental Procedures. The peptide
fragments with high-resolution data (at approximately single
amino-acid resolution) and residues 157-160 are encircled.
[0203] FIG. 96 shows color heat maps from HDX experiments across
the HLA .alpha.1 helix, the HLA .alpha.2 helix, and restricted
peptide EVDPIGHVY for all ABPs tested for HLA-PEPTIDE target G5
(HLA-B*35:01_EVDPIGHVY).
[0204] FIG. 97 shows a numerical representation of the color heat
map of FIG. 96.
[0205] FIG. 98 shows an example of data from scFv clone G5-P1C12
plotted on crystal structure of HLA-B*35:01 (5xos.pdb;
https://www.rcsb.org/structure/5XOS).
[0206] FIG. 99 shows color heat maps from a second round of HDX
experiments across the HLA .alpha.1 helix, the HLA .alpha.2 helix,
and restricted peptide AIFPGAVPAA for all ABPs tested for
HLA-PEPTIDE target G8 (HLA-A*02:01_AIFPGAVPAA).
[0207] FIG. 100 shows a numerical representation of the color heat
maps of FIG. 99.
[0208] FIG. 101 shows an example of high-resolution HDX data from
scFv G8-P1H08 plotted on a crystal structure of Fab clone G8-P1C11
complexed with HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8").
[0209] FIG. 102 shows results from a flow cytometry experiment
wherein HLA-B*35:01-transduced K562 cells were pulsed with 50 .mu.M
of target peptide EVDPIGHVY ("EVD") or negative control peptide
IPSINVHHY ("IPS"), and pHLA-specific antibodies were detected by
flow cytometry.
[0210] FIG. 103 shows results from a flow cytometry experiment
wherein HLA-A*02:01-transduced K562 cells were pulsed with 50 .mu.M
of target peptide AIFPGAVPAA ("AIF") or negative control peptide
FLLTRILTI ("FLL"), and pHLA-specific antibodies were detected by
flow cytometry.
[0211] FIG. 104 shows results from a flow cytometry experiment
wherein HLA-A*01:01-transduced K562 cells were pulsed with 50 .mu.M
of target peptide ASSLPTTMNY ("ASSL") or negative control peptide
ATDALMTGY ("ATDA"), and pHLA-specific antibodies were detected by
flow cytometry.
[0212] FIG. 105 shows BLI results for G8 target Fab clones
G8-P4F05, G8-P1B03, and G8-P5G08 to HLA-PEPTIDE target
A*02:01-AIFPGAVPAA; as well as BLI results for G5 target Fab clone
G5-P1C12 to HLA-PEPTIDE target B*35:01-EVDPIGHVY.
DETAILED DESCRIPTION
[0213] Unless otherwise defined, all terms of art, notations and
other scientific terminology used herein are intended to have the
meanings commonly understood by those of skill in the art. In some
cases, terms with commonly understood meanings are defined herein
for clarity and/or for ready reference, and the inclusion of such
definitions herein should not necessarily be construed to represent
a difference over what is generally understood in the art. The
techniques and procedures described or referenced herein are
generally well understood and commonly employed using conventional
methodologies by those skilled in the art, such as, for example,
the widely utilized molecular cloning methodologies described in
Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed.
(2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. As appropriate, procedures involving the use of commercially
available kits and reagents are generally carried out in accordance
with manufacturer-defined protocols and conditions unless otherwise
noted.
[0214] As used herein, the singular forms "a," "an," and "the"
include the plural referents unless the context clearly indicates
otherwise. The terms "include," "such as," and the like are
intended to convey inclusion without limitation, unless otherwise
specifically indicated.
[0215] As used herein, the term "comprising" also specifically
includes embodiments "consisting of" and "consisting essentially
of" the recited elements, unless specifically indicated otherwise.
For example, a multispecific ABP "comprising a diabody" includes a
multispecific ABP "consisting of a diabody" and a multispecific ABP
"consisting essentially of a diabody."
[0216] The term "about" indicates and encompasses an indicated
value and a range above and below that value. In certain
embodiments, the term "about" indicates the designated value+10%,
+5%, or +1%. In certain embodiments, where applicable, the term
"about" indicates the designated value(s).+-.one standard deviation
of that value(s).
[0217] The term "immunoglobulin" refers to a class of structurally
related proteins generally comprising two pairs of polypeptide
chains: one pair of light (L) chains and one pair of heavy (H)
chains. In an "intact immunoglobulin," all four of these chains are
interconnected by disulfide bonds. The structure of immunoglobulins
has been well characterized. See, e.g., Paul, Fundamental
Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins,
Philadelphia, Pa. Briefly, each heavy chain typically comprises a
heavy chain variable region (VH) and a heavy chain constant region
(CH). The heavy chain constant region typically comprises three
domains, abbreviated C.sub.H1, C.sub.H2, and C.sub.H3. Each light
chain typically comprises a light chain variable region (V.sub.L)
and a light chain constant region. The light chain constant region
typically comprises one domain, abbreviated C.sub.L.
[0218] The term "antigen binding protein" or "ABP" is used herein
in its broadest sense and includes certain types of molecules
comprising one or more antigen-binding domains that specifically
bind to an antigen or epitope.
[0219] In some embodiments, the ABP comprises an antibody. In some
embodiments, the ABP consists of an antibody. In some embodiments,
the ABP consists essentially of an antibody. An ABP specifically
includes intact antibodies (e.g., intact immunoglobulins), antibody
fragments, ABP fragments, and multi-specific antibodies. In some
embodiments, the ABP comprises an alternative scaffold. In some
embodiments, the ABP consists of an alternative scaffold. In some
embodiments, the ABP consists essentially of an alternative
scaffold. In some embodiments, the ABP comprises an antibody
fragment. In some embodiments, the ABP consists of an antibody
fragment. In some embodiments, the ABP consists essentially of an
antibody fragment. In some embodiments, the ABP comprises a TCR or
antigen binding portion thereof. In some embodiments, the ABP
consists of a TCR or antigen binding portion thereof. In some
embodiments, the ABP consists essentially of a TCR or antigen
binding portion thereof. In some embodiments, a CAR comprises an
ABP. An "HLA-PEPTIDE ABP," "anti-HLA-PEPTIDE ABP," or
"HLA-PEPTIDE-specific ABP" is an ABP, as provided herein, which
specifically binds to the antigen HLA-PEPTIDE. An ABP includes
proteins comprising one or more antigen-binding domains that
specifically bind to an antigen or epitope via a variable region,
such as a variable region derived from a B cell (e.g., antibody) or
T cell (e.g., TCR).
[0220] The term "antibody" herein is used in the broadest sense and
includes polyclonal and monoclonal antibodies, including intact
antibodies and functional (antigen-binding) antibody fragments,
including fragment antigen binding (Fab) fragments, F(ab')2
fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG)
fragments, variable heavy chain (VH) regions capable of
specifically binding the antigen, single chain antibody fragments,
including single chain variable fragments (scFv), and single domain
antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term
encompasses genetically engineered and/or otherwise modified forms
of immunoglobulins, such as intrabodies, peptibodies, chimeric
antibodies, fully human antibodies, humanized antibodies, and
heteroconjugate antibodies, multispecific, e.g., bispecific,
antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv,
tandem tri-scFv. Unless otherwise stated, the term "antibody"
should be understood to encompass functional antibody fragments
thereof. The term also encompasses intact or full-length
antibodies, including antibodies of any class or sub-class,
including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
[0221] As used herein, "variable region" refers to a variable
nucleotide sequence that arises from a recombination event, for
example, it can include a V, J, and/or D region of an
immunoglobulin or T cell receptor (TCR) sequence from a B cell or T
cell, such as an activated T cell or an activated B cell.
[0222] The term "antigen-binding domain" means the portion of an
ABP that is capable of specifically binding to an antigen or
epitope. One example of an antigen-binding domain is an
antigen-binding domain formed by an antibody V.sub.H-V.sub.L dimer
of an ABP. Another example of an antigen-binding domain is an
antigen-binding domain formed by diversification of certain loops
from the tenth fibronectin type III domain of an Adnectin. An
antigen-binding domain can include antibody CDRs 1, 2, and 3 from a
heavy chain in that order; and antibody CDRs 1, 2, and 3 from a
light chain in that order. An antigen-binding domain can include
TCR CDRs, e.g., .alpha.CDR1, .alpha.CDR2, .alpha.CDR3, .beta.CDR1,
.beta.CDR2, and .beta.CDR3. TCR CDRs are described herein.
[0223] The antibody V.sub.H and V.sub.L regions may be further
subdivided into regions of hypervariability ("hypervariable regions
(HVRs);" also called "complementarity determining regions" (CDRs))
interspersed with regions that are more conserved. The more
conserved regions are called framework regions (FRs). Each V.sub.H
and V.sub.L generally comprises three antibody CDRs and four FRs,
arranged in the following order (from N-terminus to C-terminus):
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The antibody CDRs are involved in
antigen binding, and influence antigen specificity and binding
affinity of the ABP. See Kabat et al., Sequences of Proteins of
Immunological Interest 5th ed. (1991) Public Health Service,
National Institutes of Health, Bethesda, Md., incorporated by
reference in its entirety.
[0224] The light chain from any vertebrate species can be assigned
to one of two types, called kappa (.kappa.) and lambda (.lamda.),
based on the sequence of its constant domain.
[0225] The heavy chain from any vertebrate species can be assigned
to one of five different classes (or isotypes): IgA, IgD, IgE, IgG,
and IgM. These classes are also designated .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively. The IgG and IgA classes
are further divided into subclasses on the basis of differences in
sequence and function. Humans express the following subclasses:
IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
[0226] The amino acid sequence boundaries of an antibody CDR can be
determined by one of skill in the art using any of a number of
known numbering schemes, including those described by Kabat et al.,
supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J Mol.
Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al.,
1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme);
Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT"
numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001,
309:657-70 ("AHo" numbering scheme); each of which is incorporated
by reference in its entirety.
[0227] Table 20 provides the positions of antibody CDR-L1, CDR-L2,
CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and
Chothia schemes. For CDR-H1, residue numbering is provided using
both the Kabat and Chothia numbering schemes.
[0228] Antibody CDRs may be assigned, for example, using ABP
numbering software, such as Abnum, available at
www.bioinf.org.uk/abs/abnum/, and described in Abhinandan and
Martin, Immunology, 2008, 45:3832-3839, incorporated by reference
in its entirety.
TABLE-US-00013 TABLE 20 Residues in CDRs according to Kabat and
Chothia numbering schemes CDR Kabat Chothia L1 L24-L34 L24-L34 L2
L50-L56 L50-L56 L3 L89-L97 L89-L97 H1 (Kabat Numbering) H31-H35B
H26-H32 or H34* H1 (Chothia Numbering) H31-H35 H26-H32 H2 H50-H65
H52-H56 H3 H95-H102 H95-H102 *The C-terminus of CDR-H1, when
numbered using the Kabat numbering convention, varies between H32
and H34, depending on the length of the CDR.
[0229] The "EU numbering scheme" is generally used when referring
to a residue in an ABP heavy chain constant region (e.g., as
reported in Kabat et al., supra). Unless stated otherwise, the EU
numbering scheme is used to refer to residues in ABP heavy chain
constant regions described herein.
[0230] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a naturally
occurring antibody structure and having heavy chains that comprise
an Fc region. For example, when used to refer to an IgG molecule, a
"full length antibody" is an antibody that comprises two heavy
chains and two light chains.
[0231] The amino acid sequence boundaries of a TCR CDR can be
determined by one of skill in the art using any of a number of
known numbering schemes, including but not limited to the IMGT
unique numbering, as described by LeFranc, M.-P, Immunol Today.
1997 November; 18(11):509; Lefranc, M.-P., "IMGT Locus on Focus: A
new section of Experimental and Clinical Immunogenetics", Exp.
Clin. Immunogenet., 15, 1-7 (1998); Lefranc and Lefranc, The T Cell
Receptor FactsBook; and M.-P. Lefranc/Developmental and Comparative
Immunology 27 (2003) 55-77, all of which are incorporated by
reference.
[0232] An "ABP fragment" comprises a portion of an intact ABP, such
as the antigen-binding or variable region of an intact ABP. ABP
fragments include, for example, Fv fragments, Fab fragments,
F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and
scFv-Fc fragments. ABP fragments include antibody fragments.
Antibody fragments can include Fv fragments, Fab fragments, F(ab')2
fragments, Fab' fragments, scFv (sFv) fragments, scFv-Fc fragments,
and TCR fragments.
[0233] "Fv" fragments comprise a non-covalently-linked dimer of one
heavy chain variable domain and one light chain variable
domain.
[0234] "Fab" fragments comprise, in addition to the heavy and light
chain variable domains, the constant domain of the light chain and
the first constant domain (CHI) of the heavy chain. Fab fragments
may be generated, for example, by recombinant methods or by papain
digestion of a full-length ABP.
[0235] "F(ab').sub.2" fragments contain two Fab' fragments joined,
near the hinge region, by disulfide bonds. F(ab').sub.2 fragments
may be generated, for example, by recombinant methods or by pepsin
digestion of an intact ABP. The F(ab') fragments can be
dissociated, for example, by treatment with
.beta.-mercaptoethanol.
[0236] "Single-chain Fv" or "sFv" or "scFv" fragments comprise a VH
domain and a VL domain in a single polypeptide chain. The VH and VL
are generally linked by a peptide linker. See Pluckthun A. (1994).
Any suitable linker may be used. In some embodiments, the linker is
a (GGGGS).sub.n. In some embodiments, n=1, 2, 3, 4, 5, or 6. See
ABPs from Escherichia coli. In Rosenberg M. & Moore G. P.
(Eds.), The Pharmacology of Monoclonal ABPs vol. 113 (pp. 269-315).
Springer-Verlag, New York, incorporated by reference in its
entirety.
[0237] "scFv-Fc" fragments comprise an scFv attached to an Fc
domain. For example, an Fc domain may be attached to the C-terminal
of the scFv. The Fc domain may follow the V.sub.H or V.sub.L,
depending on the orientation of the variable domains in the scFv
(i.e., V.sub.H-V.sub.L or V.sub.L-V.sub.H). Any suitable Fc domain
known in the art or described herein may be used. In some cases,
the Fc domain comprises an IgG4 Fc domain.
[0238] The term "single domain antibody" refers to a molecule in
which one variable domain of an ABP specifically binds to an
antigen without the presence of the other variable domain. Single
domain ABPs, and fragments thereof, are described in Arabi
Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans
et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is
incorporated by reference in its entirety. Single domain ABPs are
also known as sdAbs or nanobodies.
[0239] The term "Fe region" or "Fc" means the C-terminal region of
an immunoglobulin heavy chain that, in naturally occurring
antibodies, interacts with Fc receptors and certain proteins of the
complement system. The structures of the Fc regions of various
immunoglobulins, and the glycosylation sites contained therein, are
known in the art. See Schroeder and Cavacini, J. Allergy Clin.
Immunol., 2010, 125:S41-52, incorporated by reference in its
entirety. The Fc region may be a naturally occurring Fc region, or
an Fc region modified as described in the art or elsewhere in this
disclosure.
[0240] The term "alternative scaffold" refers to a molecule in
which one or more regions may be diversified to produce one or more
antigen-binding domains that specifically bind to an antigen or
epitope. In some embodiments, the antigen-binding domain binds the
antigen or epitope with specificity and affinity similar to that of
an ABP. Exemplary alternative scaffolds include those derived from
fibronectin (e.g., Adnectins.TM.), the .beta.-sandwich (e.g.,
iMab), lipocalin (e.g., Anticalins.RTM.), EETI-II/AGRP,
BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domains), thioredoxin peptide
aptamers, protein A (e.g., Affibody.RTM.), ankyrin repeats (e.g.,
DARPins), gamma-B-crystallin/ubiquitin (e.g., Affilins), CTLD3
(e.g., Tetranectins), Fynomers, and (LDLR-A module) (e.g.,
Avimers). Additional information on alternative scaffolds is
provided in Binz et al., Nat. Biotechnol., 2005 23:1257-1268;
Skerra, Current Opin. in Biotech., 2007 18:295-304; and Silacci et
al., J. Biol. Chem., 2014, 289:14392-14398; each of which is
incorporated by reference in its entirety. An alternative scaffold
is one type of ABP.
[0241] A "multispecific ABP" is an ABP that comprises two or more
different antigen-binding domains that collectively specifically
bind two or more different epitopes. The two or more different
epitopes may be epitopes on the same antigen (e.g., a single
HLA-PEPTIDE molecule expressed by a cell) or on different antigens
(e.g., different HLA-PEPTIDE molecules expressed by the same cell,
or a HLA-PEPTIDE molecule and a non-HLA-PEPTIDE molecule). In some
aspects, a multi-specific ABP binds two different epitopes (i.e., a
"bispecific ABP"). In some aspects, a multi-specific ABP binds
three different epitopes (i.e., a "trispecific ABP").
[0242] A "monospecific ABP" is an ABP that comprises one or more
binding sites that specifically bind to a single epitope. An
example of a monospecific ABP is a naturally occurring IgG molecule
which, while divalent (i.e., having two antigen-binding domains),
recognizes the same epitope at each of the two antigen-binding
domains. The binding specificity may be present in any suitable
valency.
[0243] The term "monoclonal antibody" refers to an antibody from a
population of substantially homogeneous antibodies. A population of
substantially homogeneous antibodies comprises antibodies that are
substantially similar and that bind the same epitope(s), except for
variants that may normally arise during production of the
monoclonal antibody. Such variants are generally present in only
minor amounts. A monoclonal antibody is typically obtained by a
process that includes the selection of a single antibody from a
plurality of antibodies. For example, the selection process can be
the selection of a unique clone from a plurality of clones, such as
a pool of hybridoma clones, phage clones, yeast clones, bacterial
clones, or other recombinant DNA clones. The selected antibody can
be further altered, for example, to improve affinity for the target
("affinity maturation"), to humanize the antibody, to improve its
production in cell culture, and/or to reduce its immunogenicity in
a subject.
[0244] The term "chimeric antibody" refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0245] "Humanized" forms of non-human antibodies are chimeric
antibodies that contain minimal sequence derived from the non-human
antibody. A humanized antibody is generally a human antibody
(recipient antibody) in which residues from one or more CDRs are
replaced by residues from one or more CDRs of a non-human antibody
(donor antibody). The donor antibody can be any suitable non-human
antibody, such as a mouse, rat, rabbit, chicken, or non-human
primate antibody having a desired specificity, affinity, or
biological effect. In some instances, selected framework region
residues of the recipient antibody are replaced by the
corresponding framework region residues from the donor antibody.
Humanized antibodies may also comprise residues that are not found
in either the recipient antibody or the donor antibody. Such
modifications may be made to further refine antibody function. For
further details, see Jones et al., Nature, 1986, 321:522-525;
Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op.
Struct. Biol., 1992, 2:593-596, each of which is incorporated by
reference in its entirety.
[0246] A "human antibody" is one which possesses an amino acid
sequence corresponding to that of an antibody produced by a human
or a human cell, or derived from a non-human source that utilizes a
human antibody repertoire or human antibody-encoding sequences
(e.g., obtained from human sources or designed de novo). Human
antibodies specifically exclude humanized antibodies.
[0247] "Affinity" refers to the strength of the sum total of
non-covalent interactions between a single binding site of a
molecule (e.g., an ABP) and its binding partner (e.g., an antigen
or epitope). Unless indicated otherwise, as used herein, "affinity"
refers to intrinsic binding affinity, which reflects a 1:1
interaction between members of a binding pair (e.g., ABP and
antigen or epitope). The affinity of a molecule X for its partner Y
can be represented by the dissociation equilibrium constant
(K.sub.D). The kinetic components that contribute to the
dissociation equilibrium constant are described in more detail
below. Affinity can be measured by common methods known in the art,
including those described herein, such as surface plasmon resonance
(SPR) technology (e.g., BIACORE.RTM.) or biolayer interferometry
(e.g., FORTEBIO.RTM.).
[0248] With regard to the binding of an ABP to a target molecule,
the terms "bind," "specific binding," "specifically binds to,"
"specific for," "selectively binds," and "selective for" a
particular antigen (e.g., a polypeptide target) or an epitope on a
particular antigen mean binding that is measurably different from a
non-specific or non-selective interaction (e.g., with a non-target
molecule). Specific binding can be measured, for example, by
measuring binding to a target molecule and comparing it to binding
to a non-target molecule. Specific binding can also be determined
by competition with a control molecule that mimics the epitope
recognized on the target molecule. In that case, specific binding
is indicated if the binding of the ABP to the target molecule is
competitively inhibited by the control molecule. In some aspects,
the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less
than about 50% of the affinity for HLA-PEPTIDE. In some aspects,
the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less
than about 40% of the affinity for HLA-PEPTIDE. In some aspects,
the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less
than about 30% of the affinity for HLA-PEPTIDE. In some aspects,
the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less
than about 20% of the affinity for HLA-PEPTIDE. In some aspects,
the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less
than about 10% of the affinity for HLA-PEPTIDE. In some aspects,
the affinity of a HLA-PEPTIDE ABP for a non-target molecule is less
than about 1% of the affinity for HLA-PEPTIDE. In some aspects, the
affinity of a HLA-PEPTIDE ABP for a non-target molecule is less
than about 0.1% of the affinity for HLA-PEPTIDE.
[0249] The term "k.sub.d" (sec.sup.-1), as used herein, refers to
the dissociation rate constant of a particular ABP-antigen
interaction. This value is also referred to as the k.sub.off
value.
[0250] The term "k.sub.d" (M.sup.-1.times.sec.sup.-1), as used
herein, refers to the association rate constant of a particular
ABP-antigen interaction. This value is also referred to as the
k.sub.on value.
[0251] The term "K.sub.D" (M), as used herein, refers to the
dissociation equilibrium constant of a particular ABP-antigen
interaction. K.sub.D=k.sub.d/k.sub.a. In some embodiments, the
affinity of an ABP is described in terms of the K.sub.D for an
interaction between such ABP and its antigen. For clarity, as known
in the art, a smaller K.sub.D value indicates a higher affinity
interaction, while a larger K.sub.D value indicates a lower
affinity interaction.
[0252] The term "K.sub.A" (M.sup.-1), as used herein, refers to the
association equilibrium constant of a particular ABP-antigen
interaction. K.sub.A=k.sub.a/k.sub.d.
[0253] An "immunoconjugate" is an ABP conjugated to one or more
heterologous molecule(s), such as a therapeutic (cytokine, for
example) or diagnostic agent.
[0254] "Fc effector functions" refer to those biological activities
mediated by the Fc region of an ABP having an Fc region, which
activities may vary depending on isotype. Examples of ABP effector
functions include C1q binding to activate complement dependent
cytotoxicity (CDC), Fc receptor binding to activate ABP-dependent
cellular cytotoxicity (ADCC), and ABP dependent cellular
phagocytosis (ADCP).
[0255] When used herein in the context of two or more ABPs, the
term "competes with" or "cross-competes with" indicates that the
two or more ABPs compete for binding to an antigen (e.g.,
HLA-PEPTIDE). In one exemplary assay, HLA-PEPTIDE is coated on a
surface and contacted with a first HLA-PEPTIDE ABP, after which a
second HLA-PEPTIDE ABP is added. In another exemplary assay, a
first HLA-PEPTIDE ABP is coated on a surface and contacted with
HLA-PEPTIDE, and then a second HLA-PEPTIDE ABP is added. If the
presence of the first HLA-PEPTIDE ABP reduces binding of the second
HLA-PEPTIDE ABP, in either assay, then the ABPs compete with each
other. The term "competes with" also includes combinations of ABPs
where one ABP reduces binding of another ABP, but where no
competition is observed when the ABPs are added in the reverse
order. However, in some embodiments, the first and second ABPs
inhibit binding of each other, regardless of the order in which
they are added. In some embodiments, one ABP reduces binding of
another ABP to its antigen by at least 25%, at least 50%, at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, or at
least 95%. A skilled artisan can select the concentrations of the
ABPs used in the competition assays based on the affinities of the
ABPs for HLA-PEPTIDE and the valency of the ABPs. The assays
described in this definition are illustrative, and a skilled
artisan can utilize any suitable assay to determine if ABPs compete
with each other. Suitable assays are described, for example, in Cox
et al., "Immunoassay Methods," in Assay Guidance Manual[Internet],
Updated Dec. 24, 2014 (www.ncbi.nlm.nih.gov/books/NBK92434/;
accessed Sep. 29, 2015); Silman et al., Cytometry, 2001, 44:30-37;
and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358; each
of which is incorporated by reference in its entirety.
[0256] The term "epitope" means a portion of an antigen that
specifically binds to an ABP. Epitopes frequently consist of
surface-accessible amino acid residues and/or sugar side chains and
may have specific three dimensional structural characteristics, as
well as specific charge characteristics. Conformational and
non-conformational epitopes are distinguished in that the binding
to the former but not the latter may be lost in the presence of
denaturing solvents. An epitope may comprise amino acid residues
that are directly involved in the binding, and other amino acid
residues, which are not directly involved in the binding. The
epitope to which an ABP binds can be determined using known
techniques for epitope determination such as, for example, testing
for ABP binding to HLA-PEPTIDE variants with different
point-mutations, or to chimeric HLA-PEPTIDE variants.
[0257] Percent "identity" between a polypeptide sequence and a
reference sequence, is defined as the percentage of amino acid
residues in the polypeptide sequence that are identical to the
amino acid residues in the reference sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared.
[0258] A "conservative substitution" or a "conservative amino acid
substitution," refers to the substitution an amino acid with a
chemically or functionally similar amino acid. Conservative
substitution tables providing similar amino acids are well known in
the art. By way of example, the groups of amino acids provided in
Tables 21-23 are, in some embodiments, considered conservative
substitutions for one another.
TABLE-US-00014 TABLE 21 Selected groups of amino acids that are
considered conservative substitutions for one another, in certain
embodiments. Acidic Residues D and E Basic Residues K, R, and H
Hydrophilic Uncharged Residues S, T, N, and Q Aliphatic Uncharged
Residues G, A, V, L, and I Non-polar Uncharged Residues C, M, and P
Aromatic Residues F, Y, and W
TABLE-US-00015 TABLE 22 Additional selected groups of amino acids
that are considered conservative substitutions for one another, in
certain embodiments. Group 1 A, S, and T Group 2 D and E Group 3 N
and Q Group 4 R and K Group 5 I, L, and M Group 6 F, Y, and W
TABLE-US-00016 TABLE 23 Further selected groups of amino acids that
are considered conservative substitutions for one another, in
certain embodiments. Group A A and G Group B D and E Group C N and
Q Group D R, K, and H Group E I, L, M, V Group F F, Y, and W Group
G S and T Group H C and M
[0259] Additional conservative substitutions may be found, for
example, in Creighton, Proteins: Structures and Molecular
Properties 2nd ed. (1993) W. H. Freeman & Co., New York, N.Y.
An ABP generated by making one or more conservative substitutions
of amino acid residues in a parent ABP is referred to as a
"conservatively modified variant."
[0260] The term "amino acid" refers to the twenty common naturally
occurring amino acids. Naturally occurring amino acids include
alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic
acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine
(Gln; Q), Glycine (Gly; G); histidine (His; H), isoleucine (Ile;
I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M),
phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S),
threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and
valine (Val; V).
[0261] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
[0262] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which an
exogenous nucleic acid has been introduced, and the progeny of such
cells. Host cells include "transformants" (or "transformed cells")
and "transfectants" (or "transfected cells"), which each include
the primary transformed or transfected cell and progeny derived
therefrom. Such progeny may not be completely identical in nucleic
acid content to a parent cell, and may contain mutations.
[0263] The term "treating" (and variations thereof such as "treat"
or "treatment") refers to clinical intervention in an attempt to
alter the natural course of a disease or condition in a subject in
need thereof. Treatment can be performed both for prophylaxis and
during the course of clinical pathology. Desirable effects of
treatment include preventing occurrence or recurrence of disease,
alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of the disease, preventing metastasis,
decreasing the rate of disease progression, amelioration or
palliation of the disease state, and remission or improved
prognosis.
[0264] As used herein, the term "therapeutically effective amount"
or "effective amount" refers to an amount of an ABP or
pharmaceutical composition provided herein that, when administered
to a subject, is effective to treat a disease or disorder.
[0265] As used herein, the term "subject" means a mammalian
subject. Exemplary subjects include humans, monkeys, dogs, cats,
mice, rats, cows, horses, camels, goats, rabbits, and sheep. In
certain embodiments, the subject is a human. In some embodiments
the subject has a disease or condition that can be treated with an
ABP provided herein. In some aspects, the disease or condition is a
cancer. In some aspects, the disease or condition is a viral
infection.
[0266] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic or
diagnostic products (e.g., kits) that contain information about the
indications, usage, dosage, administration, combination therapy,
contraindications and/or warnings concerning the use of such
therapeutic or diagnostic products.
[0267] The term "tumor" refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all pre-cancerous
and cancerous cells and tissues. The terms "cancer," "cancerous,"
"cell proliferative disorder," "proliferative disorder" and "tumor"
are not mutually exclusive as referred to herein. The terms "cell
proliferative disorder" and "proliferative disorder" refer to
disorders that are associated with some degree of abnormal cell
proliferation. In some embodiments, the cell proliferative disorder
is a cancer. In some aspects, the tumor is a solid tumor. In some
aspects, the tumor is a hematologic malignancy.
[0268] The term "pharmaceutical composition" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective
in treating a subject, and which contains no additional components
which are unacceptably toxic to the subject in the amounts provided
in the pharmaceutical composition.
[0269] The terms "modulate" and "modulation" refer to reducing or
inhibiting or, alternatively, activating or increasing, a recited
variable.
[0270] The terms "increase" and "activate" refer to an increase of
10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,
2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold,
100-fold, or greater in a recited variable.
[0271] The terms "reduce" and "inhibit" refer to a decrease of 10%,
20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold,
3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or
greater in a recited variable.
[0272] The term "agonize" refers to the activation of receptor
signaling to induce a biological response associated with
activation of the receptor. An "agonist" is an entity that binds to
and agonizes a receptor.
[0273] The term "antagonize" refers to the inhibition of receptor
signaling to inhibit a biological response associated with
activation of the receptor. An "antagonist" is an entity that binds
to and antagonizes a receptor.
[0274] The terms "nucleic acids" and "polynucleotides" may be used
interchangeably herein to refer to polymeric form of nucleotides of
any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. Polynucleotides can include, but are not limited
to coding or non-coding regions of a gene or gene fragment, loci
(locus) defined from linkage analysis, exons, introns, messenger
RNA (mRNA), cDNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA, isolated RNA,
nucleic acid probes, and primers. A polynucleotide may comprise
modified nucleotides, such as methylated nucleotides and nucleotide
analogs. Exemplary modified nucleotides include, e.g.,
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-substituted adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthioN6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.
[0275] Isolated HLA-Peptide Targets
[0276] The major histocompatibility complex (MHC) is a complex of
antigens encoded by a group of linked loci, which are collectively
termed H-2 in the mouse and HLA in humans. The two principal
classes of the MHC antigens, class I and class II, each comprise a
set of cell surface glycoproteins which play a role in determining
tissue type and transplant compatibility. In transplantation
reactions, cytotoxic T-cells (CTLs) respond mainly against class I
glycoproteins, while helper T-cells respond mainly against class II
glycoproteins.
[0277] Human major histocompatibility complex (MHC) class I
molecules, referred to interchangeably herein as HLA Class I
molecules, are expressed on the surface of nearly all cells. These
molecules function in presenting peptides which are mainly derived
from endogenously synthesized proteins to, e.g., CD8+ T cells via
an interaction with the alpha-beta T-cell receptor. The class I MHC
molecule comprises a heterodimer composed of a 46-kDa a chain which
is non-covalently associated with the 12-kDa light chain beta-2
microglobulin. The .alpha. chain generally comprises .alpha.1 and
.alpha.2 domains which form a groove for presenting an
HLA-restricted peptide, and an .alpha.3 plasma membrane-spanning
domain which interacts with the CD8 co-receptor of T-cells. FIG. 1
(prior art) depicts the general structure of a Class I HLA
molecule. Some TCRs can bind MHC class I independently of CD8
coreceptor (see, e.g., Kerry S E, Buslepp J, Cramer L A, et al.
Interplay between TCR Affinity and Necessity of Coreceptor
Ligation: High-Affinity Peptide-MHC/TCR Interaction Overcomes Lack
of CD8 Engagement. Journal of immunology (Baltimore, Md.: 1950).
2003; 171(9):4493-4503.)
[0278] Class I MHC-restricted peptides (also referred to
interchangeably herein as HLA-restricted antigens, HLA-restricted
peptides, MHC-restricted antigens, restricted peptides, or
peptides) generally bind to the heavy chain alphal-alpha2 groove
via about two or three anchor residues that interact with
corresponding binding pockets in the MHC molecule. The beta-2
microglobulin chain plays an important role in MHC class I
intracellular transport, peptide binding, and conformational
stability. For most class I molecules, the formation of a
heterotrimeric complex of the MHC class I heavy chain, peptide
(self, non-self, and/or antigenic) and beta-2 microglobulin leads
to protein maturation and export to the cell-surface.
[0279] Binding of a given HLA subtype to an HLA-restricted peptide
forms a complex with a unique and novel surface that can be
specifically recognized by an ABP such as, e.g., a TCR on a T cell
or an antibody or antigen-binding fragment thereof. HLA complexed
with an HLA-restricted peptide is referred to herein as an
HLA-PEPTIDE, a pHLA, or HLA-PEPTIDE target. In some cases the
restricted peptide is located in the .alpha.1/.alpha.2 groove of
the HLA molecule. In some cases the restricted peptide is bound to
the .alpha.1/.alpha.2 groove of the HLA molecule via about two or
three anchor residues that interact with corresponding binding
pockets in the HLA molecule.
[0280] Accordingly, provided herein are antigens comprising
HLA-PEPTIDE targets. The HLA-PEPTIDE targets may comprise a
specific HLA-restricted peptide having a defined amino acid
sequence complexed with a specific HLA subtype.
[0281] HLA-PEPTIDE targets identified herein may be useful for
cancer immunotherapy. In some embodiments, the HLA-PEPTIDE targets
identified herein are presented on the surface of a tumor cell. The
HLA-PEPTIDE targets identified herein may be expressed by tumor
cells in a human subject. The HLA-PEPTIDE targets identified herein
may be expressed by tumor cells in a population of human subjects.
For example, the HLA-PEPTIDE targets identified herein may be
shared antigens which are commonly expressed in a population of
human subjects with cancer.
[0282] The HLA-PEPTIDE targets identified herein may have a
prevalence with an individual tumor type The prevalence with an
individual tumor type may be about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,
0.6%, 0.7%, 0.8%, 0.9%, 1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,
23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,
36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,
49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, or 100%.
The prevalence with an individual tumor type may be about
0.1%-100%, 0.2-50%, 0.5-25%, or 1-10%.
[0283] Preferably, HLA-PEPTIDE targets are not generally expressed
in most normal tissues. For example, the HLA-PEPTIDE targets may in
some cases not be expressed in tissues in the Genotype-Tissue
Expression (GTEx) Project, or may in some cases be expressed only
in immune privileged or non-essential tissues. Exemplary immune
privileged or non-essential tissues include testis, minor salivary
glands, the endocervix, and the thyroid. In some cases, an
HLA-PEPTIDE target may be deemed to not be expressed on essential
tissues or non-immune privileged tissues if the median expression
of a gene from which the restricted peptide is derived is less than
0.5 RPKM (Reads Per Kilobase of transcript per Million napped
reads) across GTEx samples, if the gene is not expressed with
greater than 10 RPKM across GTEX samples, if the gene was expressed
at >=5 RPKM in no more two samples across all essential tissue
samples, or any combination thereof.
[0284] Exemplary HLA Class I Subtypes of the HLA-PEPTIDE
Targets
[0285] In humans, there are many MHC haplotypes (referred to
interchangeably herein as MHC subtypes, HLA subtypes, MHC types,
and HLA types). Exemplary HLA subtypes include, by way of example
only, HLA-A2, HLA-A1, HLA-A3, HLA-A11, HLA-A23, HLA-A24, HLA-A25,
HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33,
HLA-A34, HLA-68, HLA-B7, HLA-B8, HLA-B40, HLA-B44, HLA-B13,
HLA-B15, HLA-B-18, HLA-B27, HLA-B35, HLA-B37, HLA-B38, HLA-B39,
HLA-B45, HLA-B46, HLA-B49, HLA-B51, HLA-B54, HLA-B55, HLA-B56,
HLA-B57, HLA-B58, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05,
HLA-C*06, HLA-C*07, HLA-C*12, HLA-C*14, HLA-C*16, HLA-Cw8,
HLA-A*01:01, HLA-A*02:01, HLA-A*02:03, HLA-A*02:04, HLA-A*02:07,
HLA-A*03:01, HLA-A*03:02, HLA-A*11:01, HLA-A*23:01, HLA-A*24:02,
HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A*30:02,
HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*33:03, HLA-A*68:01,
HLA-A*68:02, HLA-B*07:02, HLA-B*08:01, HLA-B*13:02, HLA-B*15:01,
HLA-B*15:03, HLA-B*18:01, HLA-B*27:02, HLA-B*27:05, HLA-B*35:01,
HLA-B*35:03, HLA-B*37:01, HLA-B*38:01, HLA-B*39:01, HLA-B*40:01,
HLA-B*40:02, HLA-B*44:02, HLA-B*44:03, HLA-B*46:01, HLA-B*49:01,
HLA-B*51:01, HLA-B*54:01, HLA-B*55:01, HLA-B*56:01, HLA-B*57:01,
HLA-B*58:01, HLA-C*01:02, HLA-C*02:02, HLA-C*03:03, HLA-C*03:04,
HLA-C*04:01, HLA-C*05:01, HLA-C*06:02, HLA-C*07:01, HLA-C*07:02,
HLA-C*07:04, HLA-C*07:06, HLA-C*12:03, HLA-C*14:02, HLA-C*16:01,
HLA-C*16:02, HLA-C*16:04, and all subtypes thereof, including,
e.g., 4 digit, 6 digit, and 8 digit subtypes. As is known to those
skilled in the art there are allelic variants of the above HLA
types, all of which are encompassed by the present invention. A
full list of HLA Class Alleles can be found on
http://hla.alleles.org/alleles/. For example, a full list of HLA
Class I Alleles can be found on
http://hla.alleles.org/alleles/class1.html.
[0286] HLA-Restricted Peptides
[0287] The HLA-restricted peptides (referred to interchangeably
herein) as "restricted peptides" can be peptide fragments of
tumor-specific genes, e.g., cancer-specific genes. Preferably, the
cancer-specific genes are expressed in cancer samples. Genes which
are aberrantly expressed in cancer samples can be identified
through a database. Exemplary databases include, by way of example
only, The Cancer Genome Atlas (TCGA) Research Network:
http://cancergenome.nih.gov/; the International Cancer Genome
Consortium: https://dcc.icgc.org/. In some embodiments, the
cancer-specific gene has an observed expression of at least 10 RPKM
in at least 5 samples from the TCGA database. The cancer-specific
gene may have an observable bimodal distribution
[0288] The cancer-specific gene may have an observed expression of
greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 transcripts
per million (TPM) in at least one TCGA tumor tissue. In preferred
embodiments, the cancer-specific gene has an observed expression of
greater than 100 TPM in at least one TCGA tumor tissue. In some
cases, the cancer specific gene has an observed bimodal
distribution of expression across TCGA samples. Without wishing to
be bound by theory, such bimodal expression pattern is consistent
with a biological model in which there is minimal expression at
baseline in all tumor samples and higher expression in a subset of
tumors experiencing epigenetic dysregulation.
[0289] Preferably, the cancer-specific gene is not generally
expressed in most normal tissues. For example, the cancer-specific
gene may in some cases not be expressed in tissues in the
Genotype-Tissue Expression (GTEx) Project, or may in some cases be
expressed in immune privileged or non-essential tissues. Exemplary
immune privileged or non-essential tissues include testis, minor
salivary glands, the endocervix, and thyroid. In some cases, an
cancer-specific gene may be deemed to not be expressed an essential
tissues or non-immune privileged tissue if the median expression of
the cancer-specific gene is less than 0.5 RPKM (Reads Per Kilobase
of transcript per Million napped reads) across GTEx samples, if the
gene is not expressed with greater than 10 RPKM across GTEX
samples, if the gene was expressed at >=5 RPKM in no more two
samples across all essential tissue samples, or any combination
thereof.
[0290] In some embodiments, the cancer-specific gene meets the
following criteria by assessment of the GTEx: (1) median GTEx
expression in brain, heart, or lung is less than 0.1 transcripts
per million (TPM), with no one sample exceeding 5 TPM, (2) median
GTEx expression in other essential organs (excluding testis,
thyroid, minor salivary gland) is less than 2 TPM with no one
sample exceeding 10 TPM.
[0291] In some embodiments, the cancer-specific gene is not likely
expressed in immune cells generally, e.g., is not an interferon
family gene, is not an eye-related gene, not an olfactory or taste
receptor gene, and is not a gene related to the circadian cycle
(e.g., not a CLOCK, PERIOD, CRY gene)
[0292] The restricted peptide preferably may be presented on the
surface of a tumor.
[0293] The restricted peptides may have a size of about 5, about 6,
about 7, about 8, about 9, about 10, about 11, about 12, about 13,
about 14, or about 15 amino molecule residues, and any range
derivable therein. In particular embodiments, the restricted
peptide has a size of about 8, about 9, about 10, about 11, or
about 12 amino molecule residues. The restricted peptide may be
about 5-15 amino acids in length, preferably may be about 7-12
amino acids in length, or more preferably may be about 8-11 amino
acids in length.
[0294] Exemplary HLA-PEPTIDE Targets
[0295] Exemplary HLA-PEPTIDE targets are shown in Tables A, A1, and
A2. In each row of Tables A, A1, and A2 the HLA allele and
corresponding HLA-restricted peptide sequence of each complex is
shown. The peptide sequence can consist of the respective sequence
shown in any one of the rows of Tables A, A1, or A2. Alternatively
the peptide sequence can comprise the respective sequence shown in
any one of the rows of Tables A, A1, or A2. Alternatively the
peptide sequence can consist essentially of the respective sequence
shown in any one of the rows of Tables A, A1, or A2.
[0296] In some embodiments, the HLA-PEPTIDE target is a target as
shown in Table A, A1, or A2.
[0297] In some embodiments, the HLA-PEPTIDE target is a target
shown in Table A, A1, or A2, with the proviso that the isolated
HLA-PEPTIDE target is not any one of Target nos. 6364-6369,
6386-6389, 6500, 6521-6524, or 6578 and is not an HLA-PEPTIDE
target found in Table B or Table C.
[0298] In some embodiments, the HLA-restricted peptide is not from
a gene selected from WT1 or MART1.
[0299] HLA Class I molecules which do not associate with a
restricted peptide ligand are generally unstable. Accordingly, the
association of the restricted peptide with the .alpha.1/.alpha.2
groove of the HLA molecule may stabilize the non-covalent
association of the .beta.2-microglobulin subunit of the HLA subtype
with the .alpha.-subunit of the HLA subtype.
[0300] Stability of the non-covalent association of the
.beta.2-microglobulin subunit of the HLA subtype with the
.alpha.-subunit of the HLA subtype can be determined using any
suitable means. For example, such stability may be assessed by
dissolving insoluble aggregates of HLA molecules in high
concentrations of urea (e.g., about 8M urea), and determining the
ability of the HLA molecule to refold in the presence of the
restricted peptide during urea removal, e.g., urea removal by
dialysis. Such refolding approaches are described in, e.g., Proc.
Natl. Acad. Sci. USA Vol. 89, pp. 3429-3433, April 1992, hereby
incorporated by reference.
[0301] For other example, such stability may be assessed using
conditional HLA Class I ligands. Conditional HLA Class I ligands
are generally designed as short restricted peptides which stabilize
the association of the .beta.2 and a subunits of the HLA Class I
molecule by binding to the .alpha.1/.alpha.2 groove of the HLA
molecule, and which contain one or more amino acid modifications
allowing cleavage of the restricted peptide upon exposure to a
conditional stimulus. Upon cleavage of the conditional ligand, the
.beta.2 and .alpha.-subunits of the HLA molecule dissociate, unless
such conditional ligand is exchanged for a restricted peptide which
binds to the .alpha.1/.alpha.2 groove and stabilizes the HLA
molecule. Conditional ligands can be designed by introducing amino
acid modifications in either known HLA peptide ligands or in
predicted high-affinity HLA peptide ligands. For HLA alleles for
which structural information is available, water-accessibility of
side chains may also be used to select positions for introduction
of the amino acid modifications. Use of conditional HLA ligands may
be advantageous by allowing the batch preparation of stable
HLA-peptide complexes which may be used to interrogate test
restricted peptides in a high throughput manner. Conditional HLA
Class I ligands, and methods of production, are described in, e.g.,
Proc Natl Acad Sci USA. 2008 Mar. 11; 105(10): 3831-3836; Proc Natl
Acad Sci USA. 2008 Mar. 11; 105(10): 3825-3830; J Exp Med. 2018 May
7; 215(5): 1493-1504; Choo, J. A. L. et al. Bioorthogonal cleavage
and exchange of major histocompatibility complex ligands by
employing azobenzene-containing peptides. Angew Chem Int Ed Engl
53, 13390-13394 (2014); Amore, A. et al. Development of a
Hypersensitive Periodate-Cleavable Amino Acid that is Methionine-
and Disulfide-Compatible and its Application in MHC Exchange
Reagents for T Cell Characterisation. ChemBioChem 14, 123-131
(2012); Rodenko, B. et al. Class I Major Histocompatibility
Complexes Loaded by a Periodate Trigger. J Am Chem Soc 131,
12305-12313 (2009); and Chang, C. X. L. et al. Conditional ligands
for Asian HLA variants facilitate the definition of CD8+ T-cell
responses in acute and chronic viral diseases. Eur J Immunol 43,
1109-1120 (2013). These references are incorporated by reference in
their entirety.
[0302] Accordingly, in some embodiments, the ability of an
HLA-restricted peptide described herein, e.g., described in Table
A, A1, or A2, to stabilize the association of the .beta.2- and
.alpha.-subunits of the HLA molecule, is assessed by performing a
conditional ligand mediated-exchange reaction and assay for HLA
stability. HLA stability can be assayed using any suitable method,
including, e.g., mass spectrometry analysis, immunoassays (e.g.,
ELISA), size exclusion chromatography, and HLA multimer staining
followed by flow cytometry assessment of T cells.
[0303] Other exemplary methods for assessing stability of the
non-covalent association of the .beta.2-microglobulin subunit of
the HLA subtype with the .alpha.-subunit of the HLA subtype include
peptide exchange using dipeptides. Peptide exchange using
dipeptides has been described in, e.g., Proc Natl Acad Sci USA.
2013 Sep. 17, 110(38):15383-8; Proc Natl Acad Sci USA. 2015 Jan. 6,
112(1):202-7, which is hereby incorporated by reference.
[0304] Provided herein are useful antigens comprising an
HLA-PEPTIDE target. The HLA-PEPTIDE targets may comprise a specific
HLA-restricted peptide having a defined amino acid sequence
complexed with a specific HLA subtype allele.
[0305] The HLA-PEPTIDE target may be isolated and/or in
substantially pure form. For example, the HLA-PEPTIDE targets may
be isolated from their natural environment, or may be produced by
means of a technical process. In some cases, the HLA-PEPTIDE target
is provided in a form which is substantially free of other peptides
or proteins.
[0306] THE HLA-PEPTIDE targets may be presented in soluble form,
and optionally may be a recombinant HLA-PEPTIDE target complex. The
skilled artisan may use any suitable method for producing and
purifying recombinant HLA-PEPTIDE targets. Suitable methods
include, e.g., use of E. coli expression systems, insect cells, and
the like. Other methods include synthetic production, e.g., using
cell free systems. An exemplary suitable cell free system is
described in WO2017089756, which is hereby incorporated by
reference in its entirety.
[0307] Also provided herein are compositions comprising an
HLA-PEPTIDE target.
[0308] In some cases, the composition comprises an HLA-PEPTIDE
target attached to a solid support. Exemplary solid supports
include, but are not limited to, beads, wells, membranes, tubes,
columns, plates, sepharose, magnetic beads, and chips. Exemplary
solid supports are described in, e.g., Catalysts 2018, 8, 92;
doi:10.3390/cata18020092, which is hereby incorporated by reference
in its entirety.
[0309] The HLA-PEPTIDE target may be attached to the solid support
by any suitable methods known in the art. In some cases, the
HLA-PEPTIDE target is covalently attached to the solid support.
[0310] In some cases, the HLA-PEPTIDE target is attached to the
solid support by way of an affinity binding pair. Affinity binding
pairs generally involved specific interactions between two
molecules. A ligand having an affinity for its binding partner
molecule can be covalently attached to the solid support, and thus
used as bait for immobilizing Common affinity binding pairs
include, e.g., streptavidin and biotin, avidin and biotin;
polyhistidine tags with metal ions such as copper, nickel, zinc,
and cobalt; and the like.
[0311] The HLA-PEPTIDE target may comprise a detectable label.
[0312] Pharmaceutical compositions comprising HLA-PEPTIDE
targets.
[0313] The composition comprising an HLA-PEPTIDE target may be a
pharmaceutical composition. Such a composition may comprise
multiple HLA-PEPTIDE targets. Exemplary pharmaceutical compositions
are described herein. The composition may be capable of eliciting
an immune response. The composition may comprise an adjuvant.
Suitable adjuvants include, but are not limited to 1018 ISS, alum,
aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA,
dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch,
ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A,
Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide
ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system,
PLG microparticles, resiquimod, SRL172, Virosomes and other
Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan,
Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass.,
USA) which is derived from saponin, mycobacterial extracts and
synthetic bacterial cell wall mimics, and other proprietary
adjuvants such as Ribi's Detox. Quil or Superfos. Adjuvants such as
incomplete Freund's or GM-CSF are useful. Several immunological
adjuvants (e.g., MF59) specific for dendritic cells and their
preparation have been described previously (Dupuis M, et al., Cell
Immunol. 1998; 186(1):18-27; Allison A C; Dev Biol Stand. 1998;
92:3-11). Also cytokines can be used. Several cytokines have been
directly linked to influencing dendritic cell migration to lymphoid
tissues (e.g., TNF-alpha), accelerating the maturation of dendritic
cells into efficient antigen-presenting cells for T-lymphocytes
(e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589,
specifically incorporated herein by reference in its entirety) and
acting as immunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J
Immunother Emphasis Tumor Immunol. 1996 (6):414-418). HLA surface
expression and processing of intracellular proteins into peptides
to present on HLA can also be enhanced by interferon-gamma
(IFN-.gamma.). See, e.g., York I A, Goldberg A L, Mo X Y, Rock K L.
Proteolysis and class I major histocompatibility complex antigen
presentation. Immunol Rev. 1999; 172:49-66; and Rock K L, Goldberg
A L. Degradation of cell proteins and the generation of MHC class
I-presented peptides. Ann Rev Immunol. 1999; 17: 12. 739-779, which
are incorporated herein by reference in their entirety.
[0314] HLA-Peptide ABPs
[0315] Also provided herein are ABPs that specifically bind to
HLA-PEPTIDE target as disclosed herein.
[0316] The HLA-PEPTIDE target may be expressed on the surface of
any suitable target cell including a tumor cell.
[0317] The ABP can specifically bind to a human leukocyte antigen
(HLA)-PEPTIDE target, wherein the HLA-PEPTIDE target comprises an
HLA-restricted peptide complexed with an HLA Class I molecule,
wherein the HLA-restricted peptide is located in the peptide
binding groove of an .alpha.1/.alpha.2 heterodimer portion of the
HLA Class I molecule.
[0318] In some aspects, the ABP does not bind HLA class I in the
absence of HLA-restricted peptide. In some aspects, the ABP does
not bind HLA-restricted peptide in the absence of human MHC class
I. In some aspects, the ABP binds tumor cells presenting human MHC
class I being complexed with HLA-restricted peptide, optionally
wherein the HLA restricted peptide is a tumor antigen
characterizing the cancer.
[0319] An ABP can bind to each portion of an HLA-PEPTIDE complex
(i.e., HLA and peptide representing each portion of the complex),
which when bound together form a novel target and protein surface
for interaction with and binding by the ABP, distinct from a
surface presented by the peptide alone or HLA subtype alone.
Generally the novel target and protein surface formed by binding of
HLA to peptide does not exist in the absence of each portion of the
HLA-PEPTIDE complex.
[0320] An ABP can be capable of specifically binding a complex
comprising HLA and an HLA-restricted peptide (HLA-PEPTIDE), e.g.,
derived from a tumor. In some aspects, the ABP does not bind HLA in
an absence of the HLA-restricted peptide derived from the tumor. In
some aspects, the ABP does not bind the HLA-restricted peptide
derived from the tumor in an absence of HLA. In some aspects, the
ABP binds a complex comprising HLA and HLA-restricted peptide when
naturally presented on a cell such as a tumor cell.
[0321] In some embodiments, an ABP provided herein modulates
binding of HLA-PEPTIDE to one or more ligands of HLA-PEPTIDE.
[0322] The ABP may specifically bind to any one of the HLA-PEPTIDE
targets as disclosed in Table A, A1, or A2. In some embodiments,
the HLA-restricted peptide is not from a gene selected from WT1 or
MART1. In some embodiments, the ABP does not specifically bind to
any one of Target nos. 6364-6369, 6386-6389, 6500, 6521-6524, or
6578 and does not specifically bind to an HLA-PEPTIDE target found
in Table B or Table C.
[0323] In more particular embodiments, the ABP specifically binds
to an HLA-PEPTIDE target selected from any one of: HLA subtype
A*02:01 complexed with an HLA-restricted peptide comprising the
sequence LLASSILCA, HLA subtype A*01:01 complexed with an
HLA-restricted peptide comprising the sequence EVDPIGHLY, HLA
subtype B*44:02 complexed with an HLA-restricted peptide comprising
the sequence GEMSSNSTAL, HLA subtype A*02:01 complexed with an
HLA-restricted peptide comprising the sequence GVYDGEEHSV, HLA
subtype *01:01 complexed with an HLA-restricted peptide comprising
the sequence EVDPIGHVY, HLA subtype HLA-A*01:01 complexed with an
HLA-restricted peptide comprising the sequence NTDNNLAVY, HLA
subtype B*35:01 complexed with an HLA-restricted peptide comprising
the sequence EVDPIGHVY, HLA subtype A*02:01 complexed with an
HLA-restricted peptide comprising the sequence AIFPGAVPAA, and HLA
subtype A*01:01 complexed with an HLA-restricted peptide comprising
the sequence ASSLPTTMNY.
[0324] In more particular embodiments, the ABP specifically binds
to an HLA-PEPTIDE target selected from any one of: HLA subtype
A*02:01 complexed with an HLA-restricted peptide consisting
essentially of the sequence LLASSILCA, HLA subtype A*01:01
complexed with an HLA-restricted peptide consisting essentially of
the sequence EVDPIGHLY, HLA subtype B*44:02 complexed with an
HLA-restricted peptide consisting essentially of the sequence
GEMSSNSTAL, HLA subtype A*02:01 complexed with an HLA-restricted
peptide consisting essentially of the sequence GVYDGEEHSV, HLA
subtype *01:01 complexed with an HLA-restricted peptide consisting
essentially of the sequence EVDPIGHVY, HLA subtype HLA-A*01:01
complexed with an HLA-restricted peptide consisting essentially of
the sequence NTDNNLAVY, HLA subtype B*35:01 complexed with an
HLA-restricted peptide consisting essentially of the sequence
EVDPIGHVY, HLA subtype A*02:01 complexed with an HLA-restricted
peptide consisting essentially of the sequence AIFPGAVPAA, and HLA
subtype A*01:01 complexed with an HLA-restricted peptide consisting
essentially of the sequence ASSLPTTMNY.
[0325] In more particular embodiments, the ABP specifically binds
to an HLA-PEPTIDE target selected from any one of: HLA subtype
A*02:01 complexed with an HLA-restricted peptide consisting of the
sequence LLASSILCA, HLA subtype A*01:01 complexed with an
HLA-restricted peptide consisting of the sequence EVDPIGHLY, HLA
subtype B*44:02 complexed with an HLA-restricted peptide consisting
of the sequence GEMSSNSTAL, HLA subtype A*02:01 complexed with an
HLA-restricted peptide consisting of the sequence GVYDGEEHSV, HLA
subtype *01:01 complexed with an HLA-restricted peptide consisting
of the sequence EVDPIGHVY, HLA subtype HLA-A*01:01 complexed with
an HLA-restricted peptide consisting of the sequence NTDNNLAVY, HLA
subtype B*35:01 complexed with an HLA-restricted peptide consisting
of the sequence EVDPIGHVY, HLA subtype A*02:01 complexed with an
HLA-restricted peptide consisting of the sequence AIFPGAVPAA, and
HLA subtype A*01:01 complexed with an HLA-restricted peptide
consisting of the sequence ASSLPTTMNY.
[0326] In some embodiments, an ABP is an ABP that competes with an
illustrative ABP provided herein. In some aspects, the ABP that
competes with the illustrative ABP provided herein binds the same
epitope as an illustrative ABP provided herein.
[0327] In some embodiments, the ABPs described herein are referred
to herein as "variants." In some embodiments, such variants are
derived from a sequence provided herein, for example, by affinity
maturation, site directed mutagenesis, random mutagenesis, or any
other method known in the art or described herein. In some
embodiments, such variants are not derived from a sequence provided
herein and may, for example, be isolated de novo according to the
methods provided herein for obtaining ABPs. In some embodiments, a
variant is derived from any of the sequences provided herein,
wherein one or more conservative amino acid substitutions are made.
In some embodiments, a variant is derived from any of the sequences
provided herein, wherein one or more nonconservative amino acid
substitutions are made. Conservative amino acid substitutions are
described herein. Exemplary nonconservative amino acid
substitutions include those described in J Immunol. 2008 May 1;
180(9):6116-31, which is hereby incorporated by reference in its
entirety. In preferred embodiments, the non-conservative amino acid
substitution does not interfere with or inhibit the biological
activity of the functional variant. In yet more preferred
embodiments, the non-conservative amino acid substitution enhances
the biological activity of the functional variant, such that the
biological activity of the functional variant is increased as
compared to the parent ABP.
[0328] ABPs Comprising an Antibody or Antigen-Binding Fragment
Thereof
[0329] An ABP may comprise an antibody or antigen-binding fragment
thereof.
[0330] In some embodiments, the ABPs provided herein comprise a
light chain. In some aspects, the light chain is a kappa light
chain. In some aspects, the light chain is a lambda light
chain.
[0331] In some embodiments, the ABPs provided herein comprise a
heavy chain. In some aspects, the heavy chain is an IgA. In some
aspects, the heavy chain is an IgD. In some aspects, the heavy
chain is an IgE. In some aspects, the heavy chain is an IgG. In
some aspects, the heavy chain is an IgM. In some aspects, the heavy
chain is an IgG1. In some aspects, the heavy chain is an IgG2. In
some aspects, the heavy chain is an IgG3. In some aspects, the
heavy chain is an IgG4. In some aspects, the heavy chain is an
IgA1. In some aspects, the heavy chain is an IgA2.
[0332] In some embodiments, the ABPs provided herein comprise an
antibody fragment. In some embodiments, the ABPs provided herein
consist of an antibody fragment. In some embodiments, the ABPs
provided herein consist essentially of an antibody fragment. In
some aspects, the ABP fragment is an Fv fragment. In some aspects,
the ABP fragment is a Fab fragment. In some aspects, the ABP
fragment is a F(ab')2 fragment. In some aspects, the ABP fragment
is a Fab' fragment. In some aspects, the ABP fragment is an scFv
(sFv) fragment. In some aspects, the ABP fragment is an scFv-Fc
fragment. In some aspects, the ABP fragment is a fragment of a
single domain ABP.
[0333] In some embodiments, an ABP fragment provided herein is
derived from an illustrative ABP provided herein. In some
embodiments, an ABP fragments provided herein is not derived from
an illustrative ABP provided herein and may, for example, be
isolated de novo according to the methods provided herein for
obtaining ABP fragments.
[0334] In some embodiments, an ABP fragment provided herein retains
the ability to bind the HLA-PEPTIDE target, as measured by one or
more assays or biological effects described herein. In some
embodiments, an ABP fragment provided herein retains the ability to
prevent HLA-PEPTIDE from interacting with one or more of its
ligands, as described herein.
[0335] In some embodiments, the ABPs provided herein are monoclonal
ABPs. In some embodiments, the ABPs provided herein are polyclonal
ABPs.
[0336] In some embodiments, the ABPs provided herein comprise a
chimeric ABP. In some embodiments, the ABPs provided herein consist
of a chimeric ABP. In some embodiments, the ABPs provided herein
consist essentially of a chimeric ABP. In some embodiments, the
ABPs provided herein comprise a humanized ABP. In some embodiments,
the ABPs provided herein consist of a humanized ABP. In some
embodiments, the ABPs provided herein consist essentially of a
humanized ABP. In some embodiments, the ABPs provided herein
comprise a human ABP. In some embodiments, the ABPs provided herein
consist of a human ABP. In some embodiments, the ABPs provided
herein consist essentially of a human ABP.
[0337] In some embodiments, the ABPs provided herein comprise an
alternative scaffold. In some embodiments, the ABPs provided herein
consist of an alternative scaffold. In some embodiments, the ABPs
provided herein consist essentially of an alternative scaffold. Any
suitable alternative scaffold may be used. In some aspects, the
alternative scaffold is selected from an Adnectin.TM., an iMab, an
Anticalin.RTM., an EETI-II/AGRP, a Kunitz domain, a thioredoxin
peptide aptamer, an Affibody.RTM., a DARPin, an Affilin, a
Tetranectin, a Fynomer, and an Avimer.
[0338] Also disclosed herein is an isolated humanized, human, or
chimeric ABP that competes for binding to an HLA-PEPTIDE with an
ABP disclosed herein.
[0339] Also disclosed herein is an isolated humanized, human, or
chimeric ABP that binds an HLA-PEPTIDE epitope bound by an ABP
disclosed herein.
[0340] In certain aspects, an ABP comprises a human Fc region
comprising at least one modification that reduces binding to a
human Fc receptor.
[0341] It is known that when an ABP is expressed in cells, the ABP
is modified after translation. Examples of the posttranslational
modification include cleavage of lysine at the C terminus of the
heavy chain by a carboxypeptidase; modification of glutamine or
glutamic acid at the N terminus of the heavy chain and the light
chain to pyroglutamic acid by pyroglutamylation; glycosylation;
oxidation; deamidation; and glycation, and it is known that such
posttranslational modifications occur in various ABPs (See Journal
of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447,
incorporated by reference in its entirety). In some embodiments, an
ABP is an ABP or antigen-binding fragment thereof which has
undergone posttranslational modification. Examples of an ABP or
antigen-binding fragment thereof which have undergone
posttranslational modification include an ABP or antigen-binding
fragments thereof which have undergone pyroglutamylation at the N
terminus of the heavy chain variable region and/or deletion of
lysine at the C terminus of the heavy chain. It is known in the art
that such posttranslational modification due to pyroglutamylation
at the N terminus and deletion of lysine at the C terminus does not
have any influence on the activity of the ABP or fragment thereof
(Analytical Biochemistry, 2006, Vol. 348, p. 24-39, incorporated by
reference in its entirety).
Monospecific and Multispecific HLA-PEPTIDE ABPs
[0342] In some embodiments, the ABPs provided herein are
monospecific ABPs.
[0343] In some embodiments, the ABPs provided herein are
multispecific ABPs.
[0344] In some embodiments, a multispecific ABP provided herein
binds more than one antigen. In some embodiments, a multispecific
ABP binds 2 antigens. In some embodiments, a multispecific ABP
binds 3 antigens. In some embodiments, a multispecific ABP binds 4
antigens. In some embodiments, a multispecific ABP binds 5
antigens.
[0345] In some embodiments, a multispecific ABP provided herein
binds more than one epitope on a HLA-PEPTIDE antigen. In some
embodiments, a multispecific ABP binds 2 epitopes on a HLA-PEPTIDE
antigen. In some embodiments, a multispecific ABP binds 3 epitopes
on a HLA-PEPTIDE antigen.
[0346] Many multispecific ABP constructs are known in the art, and
the ABPs provided herein may be provided in the form of any
suitable multispecific suitable construct.
[0347] In some embodiments, the multispecific ABP comprises an
immunoglobulin comprising at least two different heavy chain
variable regions each paired with a common light chain variable
region (i.e., a "common light chain ABP"). The common light chain
variable region forms a distinct antigen-binding domain with each
of the two different heavy chain variable regions. See Merchant et
al., Nature Biotechnol., 1998, 16:677-681, incorporated by
reference in its entirety.
[0348] In some embodiments, the multispecific ABP comprises an
immunoglobulin comprising an ABP or fragment thereof attached to
one or more of the N- or C-termini of the heavy or light chains of
such immunoglobulin. See Coloma and Morrison, Nature Biotechnol.,
1997, 15:159-163, incorporated by reference in its entirety. In
some aspects, such ABP comprises a tetravalent bispecific ABP.
[0349] In some embodiments, the multispecific ABP comprises a
hybrid immunoglobulin comprising at least two different heavy chain
variable regions and at least two different light chain variable
regions. See Milstein and Cuello, Nature, 1983, 305:537-540; and
Staerz and Bevan, Proc. Natl. Acad. Sci. USA, 1986, 83:1453-1457;
each of which is incorporated by reference in its entirety.
[0350] In some embodiments, the multispecific ABP comprises
immunoglobulin chains with alterations to reduce the formation of
side products that do not have multispecificity. In some aspects,
the ABPs comprise one or more "knobs-into-holes" modifications as
described in U.S. Pat. No. 5,731,168, incorporated by reference in
its entirety.
[0351] In some embodiments, the multispecific ABP comprises
immunoglobulin chains with one or more electrostatic modifications
to promote the assembly of Fc hetero-multimers. See WO 2009/089004,
incorporated by reference in its entirety.
[0352] In some embodiments, the multispecific ABP comprises a
bispecific single chain molecule. See Traunecker et al., EMBO J.,
1991, 10:3655-3659; and Gruber et al., J. Immunol., 1994,
152:5368-5374; each of which is incorporated by reference in its
entirety.
[0353] In some embodiments, the multispecific ABP comprises a heavy
chain variable domain and a light chain variable domain connected
by a polypeptide linker, where the length of the linker is selected
to promote assembly of multispecific ABP with the desired
multispecificity. For example, monospecific scFvs generally form
when a heavy chain variable domain and light chain variable domain
are connected by a polypeptide linker of more than 12 amino acid
residues. See U.S. Pat. Nos. 4,946,778 and 5,132,405, each of which
is incorporated by reference in its entirety. In some embodiments,
reduction of the polypeptide linker length to less than 12 amino
acid residues prevents pairing of heavy and light chain variable
domains on the same polypeptide chain, thereby allowing pairing of
heavy and light chain variable domains from one chain with the
complementary domains on another chain. The resulting ABP therefore
has multispecificity, with the specificity of each binding site
contributed by more than one polypeptide chain. Polypeptide chains
comprising heavy and light chain variable domains that are joined
by linkers between 3 and 12 amino acid residues form predominantly
dimers (termed diabodies). With linkers between 0 and 2 amino acid
residues, trimers (termed triabodies) and tetramers (termed
tetrabodies) are favored. However, the exact type of
oligomerization appears to depend on the amino acid residue
composition and the order of the variable domain in each
polypeptide chain (e.g., V.sub.H-linker-V.sub.L vs.
V.sub.L-linker-V.sub.H), in addition to the linker length. A
skilled person can select the appropriate linker length based on
the desired multispecificity.
Fc Region and Variants
[0354] In certain embodiments, an ABP provided herein comprises an
Fc region. An Fc region can be wild-type or a variant thereof. In
certain embodiments, an ABP provided herein comprises an Fc region
with one or more amino acid substitutions, insertions, or deletions
in comparison to a naturally occurring Fc region. In some aspects,
such substitutions, insertions, or deletions yield ABP with altered
stability, glycosylation, or other characteristics. In some
aspects, such substitutions, insertions, or deletions yield a
glycosylated ABP.
[0355] A "variant Fc region" or "engineered Fc region" comprises an
amino acid sequence that differs from that of a native-sequence Fc
region by virtue of at least one amino acid modification,
preferably one or more amino acid substitution(s). Preferably, the
variant Fc region has at least one amino acid substitution compared
to a native-sequence Fc region or to the Fc region of a parent
polypeptide, e.g., from about one to about ten amino acid
substitutions, and preferably from about one to about five amino
acid substitutions in a native-sequence Fc region or in the Fc
region of the parent polypeptide. The variant Fc region herein will
preferably possess at least about 80% homology with a
native-sequence Fc region and/or with an Fc region of a parent
polypeptide, and most preferably at least about 90% homology
therewith, more preferably at least about 95% homology
therewith.
[0356] The term "Fc-region-comprising ABP" refers to an ABP that
comprises an Fc region. The C-terminal lysine (residue 447
according to the EU numbering system) of the Fc region may be
removed, for example, during purification of the ABP or by
recombinant engineering the nucleic acid encoding the ABP.
Accordingly, an ABP having an Fc region can comprise an ABP with or
without K447.
[0357] In some aspects, the Fc region of an ABP provided herein is
modified to yield an ABP with altered affinity for an Fc receptor,
or an ABP that is more immunologically inert. In some embodiments,
the ABP variants provided herein possess some, but not all,
effector functions. Such ABPs may be useful, for example, when the
half-life of the ABP is important in vivo, but when certain
effector functions (e.g., complement activation and ADCC) are
unnecessary or deleterious.
[0358] In some embodiments, the Fc region of an ABP provided herein
is a human IgG4 Fc region comprising one or more of the hinge
stabilizing mutations S228P and L235E. See Aalberse et al.,
Immunology, 2002, 105:9-19, incorporated by reference in its
entirety. In some embodiments, the IgG4 Fc region comprises one or
more of the following mutations: E233P, F234V, and L235A. See
Armour et al., Mol. Immunol., 2003, 40:585-593, incorporated by
reference in its entirety. In some embodiments, the IgG4 Fc region
comprises a deletion at position G236.
[0359] In some embodiments, the Fc region of an ABP provided herein
is a human IgG1 Fc region comprising one or more mutations to
reduce Fc receptor binding. In some aspects, the one or more
mutations are in residues selected from S228 (e.g., S228A), L234
(e.g., L234A), L235 (e.g., L235A), D265 (e.g., D265A), and N297
(e.g., N297A). In some aspects, the ABP comprises a PVA236
mutation. PVA236 means that the amino acid sequence ELLG, from
amino acid position 233 to 236 of IgG1 or EFLG of IgG4, is replaced
by PVA. See U.S. Pat. No. 9,150,641, incorporated by reference in
its entirety.
[0360] In some embodiments, the Fc region of an ABP provided herein
is modified as described in Armour et al., Eur. J. Immunol., 1999,
29:2613-2624; WO 1999/058572; and/or U.K. Pat. App. No. 98099518;
each of which is incorporated by reference in its entirety.
[0361] In some embodiments, the Fc region of an ABP provided herein
is a human IgG2 Fc region comprising one or more of mutations A330S
and P331S.
[0362] In some embodiments, the Fc region of an ABP provided herein
has an amino acid substitution at one or more positions selected
from 238, 265, 269, 270, 297, 327 and 329. See U.S. Pat. No.
6,737,056, incorporated by reference in its entirety. Such Fc
mutants include Fc mutants with substitutions at two or more of
amino acid positions 265, 269, 270, 297 and 327, including the
so-called "DANA" Fc mutant with substitution of residues 265 and
297 with alanine. See U.S. Pat. No. 7,332,581, incorporated by
reference in its entirety. In some embodiments, the ABP comprises
an alanine at amino acid position 265. In some embodiments, the ABP
comprises an alanine at amino acid position 297.
[0363] In certain embodiments, an ABP provided herein comprises an
Fc region with one or more amino acid substitutions which improve
ADCC, such as a substitution at one or more of positions 298, 333,
and 334 of the Fc region. In some embodiments, an ABP provided
herein comprises an Fc region with one or more amino acid
substitutions at positions 239, 332, and 330, as described in Lazar
et al., Proc. Natl. Acad. Sci. USA, 2006, 103:4005-4010,
incorporated by reference in its entirety.
[0364] In some embodiments, an ABP provided herein comprises one or
more alterations that improves or diminishes C1q binding and/or
CDC. See U.S. Pat. No. 6,194,551; WO 99/51642; and Idusogie et al.,
J. Immunol., 2000, 164:4178-4184; each of which is incorporated by
reference in its entirety.
[0365] In some embodiments, an ABP provided herein comprises one or
more alterations to increase half-life. ABPs with increased
half-lives and improved binding to the neonatal Fc receptor (FcRn)
are described, for example, in Hinton et al., J. Immunol., 2006,
176:346-356; and U.S. Pat. Pub. No. 2005/0014934; each of which is
incorporated by reference in its entirety. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340,
356, 360, 362, 376, 378, 380, 382, 413, 424, 428, and 434 of an
IgG. In some embodiments, the ABP comprises one or more non-Fc
modifications that extend half-life. Exemplary non-Fc modifications
that extend half-life are described in, e.g., US20170218078, which
is hereby incorporated by reference in its entirety.
[0366] In some embodiments, an ABP provided herein comprises one or
more Fc region variants as described in U.S. Pat. Nos. 7,371,826
5,648,260, and 5,624,821; Duncan and Winter, Nature, 1988,
322:738-740; and WO 94/29351; each of which is incorporated by
reference in its entirety.
[0367] Antibodies Specific for B*35:01 EVDPIGHVY (HLA-PEPTIDE
Target "G5")
[0368] In some aspects, provided herein are ABPs comprising
antibodies or antigen-binding fragments thereof that specifically
bind an HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype B*35:01 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises, consists of, or
essentially consists of the sequence EVDPIGHVY ("G5").
[0369] CDRs
[0370] The ABP specific for B*35:01_EVDPIGHVY may comprise one or
more antibody complementarity determining region (CDR) sequences,
e.g., may comprise three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3)
and three light chain CDRs (CDR-L1, CDR-L2, CDR-L3).
[0371] The ABP specific for B*35:01_EVDPIGHVY may comprise a CDR-H3
sequence. The CDR-H3 sequence may be selected from CARDGVRYYGMDVW,
CARGVRGYDRSAGYW, CASHDYGDYGEYFQHW, CARVSWYCSSTSCGVNWFDPW,
CAKVNWNDGPYFDYW, CATPTNSGYYGPYYYYGMDVW, CARDVMDVW, CAREGYGMDVW,
CARDNGVGVDYW, CARGIADSGSYYGNGRDYYYGMDVW, CARGDYYFDYW,
CARDGTRYYGMDVW, CARDVVANFDYW, CARGHSSGWYYYYGMDVW, CAKDLGSYGGYYW,
CARSWFGGFNYHYYGMDVW, CARELPIGYGMDVW, and CARGGSYYYYGMDVW.
[0372] The ABP specific for B*35:01_EVDPIGHVY may comprise a CDR-L3
sequence. The CDR-L3 sequence may be selected from CMQGLQTPITF,
CMQALQTPPTF, CQQAISFPLTF, CQQANSFPLTF, CQQANSFPLTF, CQQSYSIPLTF,
CQQTYMMPYTF, CQQSYITPWTF, CQQSYITPYTF, CQQYYTTPYTF, CQQSYSTPLTF,
CMQALQTPLTF, CQQYGSWPRTF, CQQSYSTPVTF, CMQALQTPYTF, CQQANSFPFTF,
CMQALQTPLTF, and CQQSYSTPLTF.
[0373] The ABP specific for B*35:01_EVDPIGHVY may comprise a
particular heavy chain CDR3 (CDR-H3) sequence and a particular
light chain CDR3 (CDR-L3) sequence. In some embodiments, the ABP
comprises the CDR-H3 and the CDR-L3 from the scFv designated
G5_P7_E7, G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12,
G5-P1-E05, G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06,
G5R4-P1H11, G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, or
G5R4-P4B01. CDR sequences of identified scFvs that specifically
bind B*35:01_EVDPIGHVY are shown in Table 5. For clarity, each
identified scFv hit is designated a clone name, and each row
contains the CDR sequences for that particular clone name. For
example, the scFv identified by clone name G5_P7_E7 comprises the
heavy chain CDR3 sequence CARDGVRYYGMDVW and the light chain CDR3
sequence CMQGLQTPITF.
[0374] The ABP specific for B*35:01_EVDPIGHVY may comprise all six
CDRs from the scFv designated G5_P7_E7, G5_P7_B3, G5_P7_A5,
G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05, G5-P3G01, G5-P3G08,
G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11, G5R4-P2B10, G5R4-P2H8,
G5R4-P3G05, G5R4-P4A07, or G5R4-P4B01.
[0375] VH
[0376] The ABP specific for B*35:01_EVDPIGHVY may comprise a VH
sequence. The VH sequence may be selected from
TABLE-US-00017 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGI
INPRSGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDG
VRYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSHDINWVRQAPGQGLEWMGW
MNPNSGDTGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGV
RGYDRSAGYWGQGTLVIVSS,
EVQLLESGGGLVKPGGSLRLSCAASGFSFSSYWMSWVRQAPGKGLEWISY
ISGDSGYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCASHD
YGDYGEYFQHWGQGTLVTVSS,
EVQLLQSGGGLVQPGGSLRLSCAASGFTFSNSDMNWVRQAPGKGLEWVAY
ISSGSSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVS
WYCSSTSCGVNWFDPWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNSDMNWVRQAPGKGLEWVAS
ISSSGGYINYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVN
WNDGPYFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNFGVSWLRQAPGQGLEWMGG
IIPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCATPT
NSGYYGPYYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGW
INPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDV MDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSGYLVSWVRQAPGQGLEWMGW
INPNSGGTNTAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREG
YGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYIFRNYPMHWVRQAPGQGLEWMGW
INPDSGGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDN
GVGVDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGW
MNPNIGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGI
ADSGSYYGNGRDYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGW
INPNSGVTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGD
YYFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGW
INPNSGDTKYSQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDG
TRYYGMDVWGQGTTVTVSS,
EVQLLESGGGLVKPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSY
ISSSSSYTNYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDV
VANFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGW
MNPDSGSTGYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGH
SSGWYYYYGMDVWGQGTTVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFTSYSMHWVRQAPGKGLEWVSS
ITSFTNTMYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDL
GSYGGYYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGI
INPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSW
FGGFNYHYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGW
MNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREL
PIGYGMDVWGQGTTVTVSS, and
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
IIPIVGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGG
SYYYYGMDVWGQGTTVTVSS.
[0377] VL
[0378] The ABP specific for B*35:01_EVDPIGHVY may comprise a VL
sequence. The VL sequence may be selected from
TABLE-US-00018 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTP ITFGQGTRLEIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSSRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP PTFGPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAISFPLTFGQ STKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYS
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYA
ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTFGG GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYY
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYMMPYTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYGAS
SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPWTFGQGT KVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYITPYTFGQ GTKLEIK,
DIVMTQSPDSLAVSLGERATINCKTSQSVLYRPNNENYLAWYQQKPGQPP
KLLIYQASIREPGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTT PYTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRFLNWYQQKPGKAPKLLIYG
ASRPQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQ GTKVEIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSHRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP LTFGGGTKVEIK,
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYA
ASARASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSWPRTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKWYGAS
RLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPVTFGQGT KVEIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP YTFGQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASEDISNHLNWYQQKPGKAPKLLIYD
ALSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGP GTKVDIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP LTFGQGTKVEIK,
and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGG GTKVEIK.
[0379] VH-VL Combinations
[0380] The ABP specific for B*35:01_EVDPIGHVY may comprise a
particular VH sequence and a particular VL sequence. In some
embodiments, the ABP specific for B*35:01_EVDPIGHVY comprises a VH
sequence and VL sequence from the scFv designated G5_P7_E7,
G5_P7_B3, G5_P7_A5, G5_P7_F6, G5-P1B12, G5-P1C12, G5-P1-E05,
G5-P3G01, G5-P3G08, G5-P4B02, G5-P4E04, G5R4-P1D06, G5R4-P1H11,
G5R4-P2B10, G5R4-P2H8, G5R4-P3G05, G5R4-P4A07, or G5R4-P4B01. The
VH and VL sequences of identified scFvs that specifically bind
B*35:01_EVDPIGHVY are shown in Table 4. For clarity, each
identified scFv hit is designated a clone name, and each row
contains the VH and VL sequences for that particular clone name.
For example, the scFv identified by clone name G5_P7_E7 comprises
the VH sequence
TABLE-US-00019 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDMWVRQAPGQGLEWMGII
NPRSGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGV
RYYGMDVWGQGTTVTVSS
and the VL sequence
TABLE-US-00020 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTP
ITFGQGTRLEIK.
[0381] Antibodies Specific for a*02:01 AIFPGAVPAA (HLA-PEPTIDE
Target "G8")
[0382] In some aspects, provided herein are ABPs comprising
antibodies or antigen-binding fragments thereof that specifically
bind an HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype A*02:01 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises, consists of, or
essentially consists of the sequence AIFPGAVPAA ("G8").
[0383] CDRs
[0384] The ABP specific for A*02:01_AIFPGAVPAA may comprise one or
more antibody complementarity determining region (CDR) sequences,
e.g., may comprise three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3)
and three light chain CDRs (CDR-L1, CDR-L2, CDR-L3).
[0385] The ABP specific for A*02:01_AIFPGAVPAA may comprise a
CDR-H3 sequence. The CDR-H3 sequence may be selected from
CARDDYGDYVAYFQHW, CARDLSYYYGMDVW, CARVYDFWSVLSGFDIW,
CARVEQGYDIYYYYYMDVW, CARSYDYGDYLNFDYW, CARASGSGYYYYYGMDVW,
CAASTWIQPFDYW, CASNGNYYGSGSYYNYW, CARAVYYDFWSGPFDYW,
CAKGGIYYGSGSYPSW, CARGLYYMDVW, CARGLYGDYFLYYGMDVW,
CARGLLGFGEFLTYGMDVW, CARDRDSSWTYYYYGMDVW, CARGLYGDYFLYYGMDVW,
CARGDYYDSSGYYFPVYFDYW, and CAKDPFWSGHYYYYGMDVW.
[0386] The ABP specific for A*02:01_AIFPGAVPAA may comprise a
CDR-L3 sequence. The CDR-L3 sequence may be selected from
CQQNYNSVTF, CQQSYNTPWTF, CGQSYSTPPTF, CQQSYSAPYTF, CQQSYSIPPTF,
CQQSYSAPYTF, CQQHNSYPPTF, CQQYSTYPITI, CQQANSFPWTF, CQQSHSTPQTF,
CQQSYSTPLTF, CQQSYSTPLTF, CQQTYSTPWTF, CQQYGSSPYTF, CQQSHSTPLTF,
CQQANGFPLTF, and CQQSYSTPLTF.
[0387] The ABP specific for A*02:01_AIFPGAVPAA may comprise a
particular heavy chain CDR3 (CDR-H3) sequence and a particular
light chain CDR3 (CDR-L3) sequence. In some embodiments, the ABP
comprises the CDR-H3 and the CDR-L3 from the scFv designated
G8-P1A03, G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02,
G8-P1H08, G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05,
R3G8-P5C03, R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11. CDR
sequences of identified scFvs that specifically bind
A*02:01_AIFPGAVPAA are shown in Table 7. For clarity, each
identified scFv hit is designated a clone name, and each row
contains the CDR sequences for that particular clone name. For
example, the scFv identified by clone name G8-P1A03 comprises the
heavy chain CDR3 sequence CARDDYGDYVAYFQHW and the light chain CDR3
sequence CQQNYNSVTF.
[0388] The ABP specific for A*02:01_AIFPGAVPAA may comprise all six
CDRs from the scFv designated G8-P1A03, G8-P1A04, G8-P1A06,
G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08, G8-P2B05, G8-P2E06,
R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03, R3G8-P5F02,
R3G8-P5G08, G8-P1C01, or G8-P2C11.
[0389] VH
[0390] The ABP specific for A*02:01_AIFPGAVPAA may comprise a VH
sequence. The VH sequence may be selected from
TABLE-US-00021 QVQLVQSGAEVKKPGASVKVSCKASGGTFSRSAITWVRQAPGQGLEWMG
WINPNSGATNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DDYGDYVAYFQHWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYPFIGQYLHWVRQAPGQGLEWMG
IINPSGDSATYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DLSYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMG
WMNPIGGGTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
VYDFWSVLSGFDIWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVS
GINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
VEQGYDIYYYYYMDVWGKGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTLSSYPINWVRQAPGQGLEWMG
WISTYSGHADYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
SYDYGDYLNFDYWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVS
SISGRGDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
ASGSGYYYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYFMHWVRQAPGQGLEWMG
MVNPSGGSETFAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAA
STWIQPFDYWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFDFSIYSMNWVRQAPGKGLEWVS
AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAS
NGNYYGSGSYYNYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTLTTYYMHWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
AVYYDFWSGPFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG
WINPYSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAK
GGIYYGSGSYPSWGQGTLVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGVSWVRQAPGQGLEWMG
WISPYSGNTDYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR
GLYYMDVWGKGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSNMYLHWVRQAPGQGLEWMG
WINPNTGDTNYAQTFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GLYGDYFLYYGMDVWGQGTKVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG
WMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GLLGFGEFLTYGMDVWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYTHWVRQAPGQGLEWMG
VINPSGGSTTYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DRDSSWTYYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSNYMHWVRQAPGQGLEWMG
WMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GLYGDYFLYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSHAISWVRQAPGQGLEWMG
VIIPSGGTSYTQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARG
DYYDSSGYYFPVYFDYWGQGTLVTVSS, and
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMG
WINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DPFWSGHYYYYGMDVWGQGTTVTVSS.
[0391] VL
[0392] The ABP specific for A*02:01_AIFPGAVPAA may comprise a VL
sequence. The VL sequence may be selected from
TABLE-US-00022 DIQMTQSPSSLSASVGDRVTITCRASQSITSYLNWYQQKPGKAPKLLIY
DASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYNSVTFG QGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCWASQGISSYLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPWTF GPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQAISNSLAWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSTPPTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
KASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTF GPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPPTF GGGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTF GGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGINSYLAWYQQKPGKAPKLLIY
DASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNSYPPTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTYPITI GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNSLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQDVSTWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPQTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY
DASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTF GGGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTF GGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPWTF GQGTKLEIK,
EIVMTQSPATLSVSPGERATLSCRASQSVGNSLAWYQQKPGQAPRLLIY
GASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYGSSPYTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSHSTPLTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQNIYTYLNWYQQKPGKAPKLLIY
DASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPLTF GGGTKVEIK, and
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTF GGGTKVEIK.
[0393] VH-VL Combinations
[0394] The ABP specific for A*02:01_AIFPGAVPAA may comprise a
particular VH sequence and a particular VL sequence. In some
embodiments, the ABP specific for A*02:01_AIFPGAVPAA comprises a VH
sequence and VL sequence from the scFv designated G8-P1A03,
G8-P1A04, G8-P1A06, G8-P1B03, G8-P1C11, G8-P1D02, G8-P1H08,
G8-P2B05, G8-P2E06, R3G8-P2C10, R3G8-P2E04, R3G8-P4F05, R3G8-P5C03,
R3G8-P5F02, R3G8-P5G08, G8-P1C01, or G8-P2C11. The VH and VL
sequences of identified scFvs that specifically bind
A*02:01_AIFPGAVPAA are shown in Table 6. For clarity, each
identified scFv hit is designated a clone name, and each row
contains the VH and VL sequences for that particular clone name.
For example, the scFv identified by clone name G8-P1A03 comprises
the VH sequence
QVQLVQSGAEVKKPGASVKVSCKASGGTFSRSAITWVRQAPGQGLEWMGWINPNS
GATNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDDYGDYVAYFQH WGQGTLVTVSS
and the VL sequence
TABLE-US-00023 DIQMTQSPSSLSASVGDRVTITCRASQSITSYLNWYQQKPGKAPKWYDAS
NLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYNSVTFGQGTK LEIK.
[0395] Antibodies Specific for A*01:01 ASSLPTTMNY (HLA-PEPTIDE
Target "G10")
[0396] In some aspects, provided herein are ABPs comprising
antibodies or antigen-binding fragments thereof that specifically
bind an HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype A*01:01 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises, consists of, or
essentially consists of the sequence ASSLPTTMNY ("G10").
[0397] CDRs
[0398] The ABP specific for A*01:01_ASSLPTTMNY may comprise one or
more antibody complementarity determining region (CDR) sequences,
e.g., may comprise three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3)
and three light chain CDRs (CDR-L1, CDR-L2, CDR-L3).
[0399] The ABP specific for A*01:01_ASSLPTTMNY may comprise a
CDR-H3 sequence. The CDR-H3 sequence may be selected from
CARDQDTIFGVVITWFDPW, CARDKVYGDGFDPW, CAREDDSMDVW, CARDSSGLDPW,
CARGVGNLDYW, CARDAHQYYDFWSGYYSGTYYYGMDVW, CAREQWPSYWYFDLW,
CARDRGYSYGYFDYW, CARGSGDPNYYYYYGLDVW, CARDTGDHFDYW, CARAENGMDVW,
CARDPGGYMDVW, CARDGDAFDIW, CARDMGDAFDIW, CAREEDGMDVW, CARDTGDHFDYW,
CARGEYSSGFFFVGWFDLW, and CARETGDDAFDIW.
[0400] The ABP specific for A*01:01_ASSLPTTMNY may comprise a
CDR-L3 sequence. The CDR-L3 sequence may be selected from
CQQYFTTPYTF, CQQAEAFPYTF, CQQSYSTPITF, CQQSYIIPYTF, CHQTYSTPLTF,
CQQAYSFPWTF, CQQGYSTPLTF, CQQANSFPRTF, CQQANSLPYTF, CQQSYSTPFTF,
CQQSYSTPFTF, CQQSYGVPTF, CQQSYSTPLTF, CQQSYSTPLTF, CQQYYSYPWTF,
CQQSYSTPFTF, CMQTLKTPLSF, and CQQSYSTPLTF.
[0401] The ABP specific for A*01:01_ASSLPTTMNY may comprise a
particular heavy chain CDR3 (CDR-H3) sequence and a particular
light chain CDR3 (CDR-L3) sequence. In some embodiments, the ABP
comprises the CDR-H3 and the CDR-L3 from the scFv designated
R3G10-P1A07, R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01,
R3G10-P1H08, R3G10-P2C04, R3G10-P2G11, R3G10-P3E04, R3G10-P4A02,
R3G10-P4C05, R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12,
R3G10-P4G06, R3G10-P5A08, or R3G10-P5C08. CDR sequences of
identified scFvs that specifically bind A*01:01_ASSLPTTMNY are
shown in Table 9. For clarity, each identified scFv hit is
designated a clone name, and each row contains the CDR sequences
for that particular clone name. For example, the scFv identified by
clone name R3G10-P1A07 comprises the heavy chain CDR3 sequence
CARDQDTIFGVVITWFDPW and the light chain CDR3 sequence
CQQYFTTPYTF.
[0402] The ABP specific for A*01:01_ASSLPTTMNY may comprise all six
CDRs from the scFv designated R3G10-P1A07, R3G10-P1B07,
R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08, R3G10-P2C04,
R3G10-P2G11, R3G10-P3E04, R3G10-P4A02, R3G10-P4C05, R3G10-P4D04,
R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06, R3G10-P5A08, or
R3G10-P5C08.
[0403] VH
[0404] The ABP specific for A*01:01_ASSLPTTMNY may comprise a VH
sequence. The VH sequence may be selected from
TABLE-US-00024 EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVS
GISARSGRTYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
DQDTIFGVVITWFDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG
IIHPGGGTTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DKVYGDGFDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYIFTGYYMHWVRQAPGQGLEWMG
MIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
EDDSMDVWGKGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFIGYYMHWVRQAPGQGLEWMG
MIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DSSGLDPWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMG
MIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GVGNLDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGVTFSTSAISWVRQAPGQGLEWMG
WISPYNGNTDYAQMLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DAHQYYDFWSGYYSGTYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSNSIINWVRQAPGQGLEWMG
WMNPNSGNTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
EQWPSYWYFDLWGRGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSTHDINWVRQAPGQGLEWMG
VINPSGGSAIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DRGYSYGYFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGNTFIGYYVHWVRQAPGQGLEWVG
IINPNGGSISYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
GSGDPNYYYYYGLDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTLSYYYMHWVRQAPGQGLEWMG
MIGPSDGSTSYAQRFQGRVTMTRDTSTGTVYMELSSLRSEDTAVYYCAR
DTGDHFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMG
IIGPSDGSTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
AENGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYVHWVRQAPGQGLEWMG
IIAPSDGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DPGGYMDVWGKGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYLHWVRQAPGQGLEWMG
MIGPSDGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DGDAFDIWGQGTMVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMG
RISPSDGSTTYAPKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR
DMGDAFDIWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMG
MIGPSDGSTSYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
EEDGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTLSYYYMHWVRQAPGQGLEWMG
MIGPSDGSTSYAQRFQGRVTMTRDTSTGTVYMELSSLRSEDTAVYYCAR
DTGDHFDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASGGTFNNFAISWVRQAPGQGLEWMG
GIIPIFDATNYAQKFQGRVTFTADESTSTAYMELSSLRSEDTAVYYCAR
GEYSSGFFFVGWFDLWGRGTQVTVSS, and
QVQLVQSGAEVKKPGASVKVSCKASGYNFTGYYMHWVRQAPGQGLEWMG
IIAPSDGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
ETGDDAFDIWGQGTMVTVSS.
[0405] VL
[0406] The ABP specific for A*01:01_ASSLPTTMNY may comprise a VL
sequence. The VL sequence may be selected from
TABLE-US-00025 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIY
AASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYFTTPYTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIF
DASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAEAFPYTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITF GQGTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIY
KASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYIIPYTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQTYSTPLTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIY
SASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSFPWTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQNISSYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYSTPLTF GQGTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQDISRYLAWYQQKPGKAPKLLIY
DASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPRTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY
AASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSLPYTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
AASTLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTF GPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQRISSYLNWYQQKPGKAPKLLIY
SASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTF GPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIY
DASKLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGVPTFG QGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIY
DASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTF GGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTF GGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKLLIY
DASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSYPWTF GQGTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGSISSYLNWYQQKPGKAPKLLI
YAASTLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFT FGPGTKVDIK,
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSP
QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQTLK TPLSFGGGTKVEIK,
and DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTF GGGTKVEIK.
[0407] VH-VL Combinations
[0408] The ABP specific for A*01:01_ASSLPTTMNY may comprise a
particular VH sequence and a particular VL sequence. In some
embodiments, the ABP specific for A*01:01_ASSLPTTMNY comprises a VH
sequence and VL sequence from the scFv designated R3G10-P1A07,
R3G10-P1B07, R3G10-P1E12, R3G10-P1F06, R3G10-P1H01, R3G10-P1H08,
R3G10-P2C04, R3G10-P2G11, R3G10-P3E04, R3G10-P4A02, R3G10-P4C05,
R3G10-P4D04, R3G10-P4D10, R3G10-P4E07, R3G10-P4E12, R3G10-P4G06,
R3G10-P5A08, or R3G10-P5C08. The VH and VL sequences of identified
scFvs that specifically bind A*01:01_ASSLPTTMNY are shown in Table
8. For clarity, each identified scFv hit is designated a clone
name, and each row contains the VH and VL sequences for that
particular clone name. For example, the scFv identified by clone
name R3G10-P1A07 comprises the VH sequence
EVQLLESGGGLVKPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVSGISARSG
RTYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARDQDTIFGVVITWFDP
WGQGTLVTVSS and the VL sequence
TABLE-US-00026 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIYA
ASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYFTTPYTFGQ GTKLEIK.
[0409] Antibodies Specific for A*02:01 LLASSILCA (G7)
[0410] In some aspects, provided herein are ABPs comprising
antibodies or antigen-binding fragments thereof that specifically
bind an HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype A*02:01 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises, consists of, or
consists essentially of the sequence LLASSILCA ("G7").
[0411] Sequences of G7-Specific Antibodies
[0412] The ABP specific for A*02:01_LLASSILCA may comprise one or
more sequences, as described in further detail.
[0413] CDRs
[0414] The ABP specific for A*02:01_LLASSILCA may comprise one or
more antibody complementarity determining region (CDR) sequences,
e.g., may comprise three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3)
and three light chain CDRs (CDR-L1, CDR-L2, CDR-L3).
[0415] The ABP specific for A*02:01_LLASSILCA may comprise a CDR-H3
sequence. The CDR-H3 sequence may be selected from
CARDGYDFWSGYTSDDYW, CASDYGDYR, CARDLMTTVVTPGDYGMDVW,
CARQDGGAFAFDIW, CARELGYYYGMDVW, CARALIFGVPLLPYGMDVW,
CAKDLATVGEPYYYYGMDVW, and CARLWFGELHYYYYYGMDVW.
[0416] The ABP specific for A*02:01_LLASSILCA may comprise a CDR-L3
sequence. The CDR-L3 sequence may be selected from CHHYGRSHTF,
CQQANAFPPTF, CQQYYSIPLTF, CQQSYSTPPTF, CQQSYSFPYTF, CMQALQTPLTF,
CQQGNTFPLTF, and CMQGSHWPPSF.
[0417] The ABP specific for A*02:01_LLASSILCA may comprise a
particular heavy chain CDR3 (CDR-H3) sequence and a particular
light chain CDR3 (CDR-L3) sequence. In some embodiments, the ABP
comprises the CDR-H3 and the CDR-L3 from the scFv designated
G7R3-P1C6, G7R3-P1G10, 1-G7R3-P1B4, 2-G7R4-P2C2, 3-G7R4-P1A3,
4-G7R4-B5-P2E9, 5-G7R4-B10-P1F8, or B7 (G7R3-P3A9). CDR sequences
of identified scFvs that specifically bind A*02:01_LLASSILCA are
shown in Table 36. For clarity, each identified scFv hit is
designated a clone name, and each row contains the CDR sequences
for that particular clone name. For example, the scFv identified by
clone name G7R3-P1C6 comprises the heavy chain CDR3 sequence
CARDGYDFWSGYTSDDYW and the light chain CDR3 sequence
CHHYGRSHTF.
[0418] The ABP specific for A*02:01_LLASSILCA may comprise all six
CDRs from the scFv designated G7R3-P1C6, G7R3-P1G10, 1-G7R3-P1B4,
2-G7R4-P2C2, 3-G7R4-P1A3, 4-G7R4-B5-P2E9, 5-G7R4-B10-P1F8, or B7
(G7R3-P3A9).
[0419] VL
[0420] The ABP specific for *02:01_LLASSILCA may comprise a VL
sequence. The VL sequence may be selected from
TABLE-US-00027 EIVMTQSPATLSVSPGERATLSCRASQSVSSSNLAWYQQKPGQAPRLLI
YGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCHHYGRSHTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANAFPPTF GQGTKVEIK,
DIVMTQSPDSLAVSLGERATINCKSSQSVFYSSNNKNQLAWYQQKPGQP
PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYY SIPLTFGQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDIFKYLNWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTF GQGTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIY
YASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPYTF GQGTKVEIK,
DIVMTQSPLSLPVTPGEPASISCSSSQSLLHSNGYNYLDWYLQKPGQSP
QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQ TPLTFGGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY
SASNLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTFPLTF GQGTKVEIK, and
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSP
QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSH
WPPSFGQGTRLEIK.
[0421] VH
[0422] The ABP specific for *02:01_LLASSILCA may comprise a VH
sequence. The VH sequence may be selected from
TABLE-US-00028 QVQLVQSGAEVKKPGASVKVSCKASGGTFSNYGISWVRQAPGQGLEWMG
IINPGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARD
GYDFWSGYTSDDYWGQGTLVTVSS,
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVS
GISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAS DYGDYRGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYIHWVRQAPGQGLEWMG
WLNPNSGNTGYAQRFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
DLMTTVVTPGDYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGASMKVSCKASGYTFTTDGISWVRQAPGQGLEWMG
RIYPHSGYTEYAKKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
QDGGAFAFDIWGQGTMVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSQYMHWVRQAPGQGLEWMG
WISPNNGDTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
ELGYYYGMDVWGQGTTVTVSS,
QVQLVQSGAEVKKPGSSVKVSCKASRYTFTSYDINWVRQAPGQGLEWMG
RIIPMLNIANYAPKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR
ALIFGVPLLPYGMDVWGQGTTVTVSS,
EVQLLQSGGGLVQPGGSLRLSCAASGFTFSSSWMHWVRQAPGKGLEWVS
FISTSSGYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
DLATVGEPYYYYGMDVWGQGTTVTVSS, and
QVQLVQSGAEVKKPGSSVKVSCKASGDTFNTYALSWVRQAPGQGLEWMG
WMNPNSGNAGYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR
LWFGELHYYYYYGMDVWGQGTMVTVSS.
[0423] VH-VL Combinations
[0424] The ABP specific for A*02:01_LLASSILCA may comprise a
particular VH sequence and a particular VL sequence. In some
embodiments, the ABP specific for A*02:01_LLASSILCA comprises a VH
sequence and a VL sequence from the scFv designated G7R3-P1C6,
G7R3-P1G10, 1-G7R3-P1B4, 2-G7R4-P2C2, 3-G7R4-P1A3, 4-G7R4-B5-P2E9,
5-G7R4-B10-P1F8, or B7 (G7R3-P3A9). The VH and VL sequences of
identified scFvs that specifically bind A*02:01_LLASSILCA are shown
in Table 35. For clarity, each identified scFv hit is designated a
clone name, and each row contains the VH and VL sequences for that
particular clone name. For example, the scFv identified by clone
name G7R3-P1C6 comprises the VH sequence
QVQLVQSGAEVKKPGASVKVSCKASGGTFSNYGISWVRQAPGQGLEWMGIINPGGS
TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGYDFWSGYTSDDY WGQGTLVTVSS
and the VL sequence
TABLE-US-00029 EIVMTQSPATLSVSPGERATLSCRASQSVSSSNLAWYQQKPGQAPRLLIY
GASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCHHYGRSHTFGQ GTKVEIK.
[0425] Antibodies Specific for A*01:01 NTDNNLAVY (G2)
[0426] In some aspects, provided herein are ABPs comprising
antibodies or antigen-binding fragments thereof that specifically
bind an HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype A*01:01 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises, consists of, or
consists essentially of the sequence NTDNNLAVY ("G2").
[0427] Sequences of G2-Specific Antibodies
[0428] The ABP specific for A*01:01_NTDNNLAVY may comprise one or
more sequences, as described in further detail.
[0429] CDRs
[0430] The ABP specific for A*01:01_NTDNNLAVY may comprise one or
more antibody complementarity determining region (CDR) sequences,
e.g., may comprise three heavy chain CDRs (CDR-H1, CDR-H2, CDR-H3)
and three light chain CDRs (CDR-L1, CDR-L2, CDR-L3).
[0431] The ABP specific for A*01:01_NTDNNLAVY may comprise a CDR-H3
sequence. The CDR-H3 sequence may be selected from CAATEWLGVW,
CARANWLDYW, CARANWLDYW, CARDWVLDYW, CARGEWLDYW, CARGWELGYW,
CARDFVGYDDW, CARDYGDLDYW, CARGSYGMDVW, CARDGYSGLDVW, CARDSGVGMDVW,
CARDGVAVASDYW, CARGVNVDDFDYW, CARGDYTGNWYFDLW, CARANWLDYW,
CARDQFYGGNSGGHDYW, CAREEDYW, CARGDWFDPW, CARGDWFDPW, CARGEWFDPW,
CARSDWFDPW, CARDSGSYFDYW, CARDYGGYVDYW, CAREGPAALDVW, CARERRSGMDVW,
CARVLQEGMDVW, CASERELPFDIW, CAKGGGGYGMDVW, CAAMGIAVAGGMDVW,
CARNWNLDYW, CATYDDGMDVW, CARGGGGALDYW, CALSGNYYGMDVW,
CARGNPWELRLDYW, and CARDKNYYGMDVW.
[0432] The ABP specific for A*01:01_NTDNNLAVY may comprise a CDR-L3
sequence. The CDR-L3 sequence may be selected from CQQSYNTPYTF,
CQQSYSTPYTF, CQQSYSTPYSF, CQQSYSTPFTF, CQQSYGVPYTF, CQQSYSAPYTF,
CQQSYSAPYTF, CQQSYSAPYSF, CQQSYSTPYTF, CQQSYSVPYSF, CQQSYSAPYTF,
CQQSYSVPYSF, CQQSYSTPQTF, CQQLDSYPFTF, CQQSYSSPYTF, CQQSYSTPLTF,
CQQSYSTPYSF, CQQSYSTPYTF, CQQSYSTPYTF, CQQSYSTPFTF, CQQSYSTPTF,
CQQTYAIPLTF, CQQSYSTPYTF, CQQSYIAPFTF, CQQSYSIPLTF, CQQSYSNPTF,
CQQSYSTPYSF, CQQSYSDQWTF, CQQSYLPPYSF, CQQSYSSPYTF, CQQSYTTPWTF,
CQQSYLPPYSF, CQEGITYTF, CQQYYSYPFTF, and CQHYGYSPVTF.
[0433] The ABP specific for A*01:01_NTDNNLAVY may comprise a
particular heavy chain CDR3 (CDR-H3) sequence and a particular
light chain CDR3 (CDR-L3) sequence. In some embodiments, the ABP
comprises the CDR-H3 and the CDR-L3 from the scFv designated
G2-P2E07, G2-P2E03, G2-P2A11, G2-P2C06, G2-P1G01, G2-P1C02,
G2-P1H01, G2-P1B12, G2-P1B06, G2-P2H10, G2-P1H10, G2-P2C11,
G2-P1C09, G2-P1A10, G2-P1B10, G2-P1D07, G2-P1E05, G2-P1D03,
G2-P1G12, G2-P2H11, G2-P1C03, G2-P1G07, G2-P1F12, G2-P1G03,
G2-P2B08, G2-P2A10, G2-P2D04, G2-P1C06, G2-P2A09, G2-P1B08,
G2-P1E03, G2-P2A03, G2-P2F01, G2-P1H11, or G2-P1D06. CDR sequences
of identified scFvs that specifically bind A*01:01_NTDNNLAVY are
found in Table 34. For clarity, each identified scFv hit is
designated a clone name, and each row contains the CDR sequences
for that particular clone name. For example, the scFv identified by
clone name G2-P2E07 comprises the heavy chain CDR3 sequence
CAATEWLGVW and the light chain CDR3 sequence CQQSYNTPYTF.
[0434] The ABP specific for A*01:01_NTDNNLAVY may comprise all six
CDRs from the scFv designated G2-P2E07, G2-P2E03, G2-P2A11,
G2-P2C06, G2-P1G01, G2-P1C02, G2-P1H01, G2-P1B12, G2-P1B06,
G2-P2H10, G2-P1H10, G2-P2C11, G2-P1C09, G2-P1A10, G2-P1B10,
G2-P1D07, G2-P1E05, G2-P1D03, G2-P1G12, G2-P2H11, G2-P1C03,
G2-P1G07, G2-P1F12, G2-P1G03, G2-P2B08, G2-P2A10, G2-P2D04,
G2-P1C06, G2-P2A09, G2-P1B08, G2-P1E03, G2-P2A03, G2-P2F01,
G2-P1H11, or G2-P1D06.
[0435] VL
[0436] The ABP specific for A*01:01_NTDNNLAVY may comprise a VL
sequence. The VL sequence may be selected from
TABLE-US-00030 DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIY
AASSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPYTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIY
AASTVQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQDISRWLAWYQQKPGKAPKLLIY
AASRLQAGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYSF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQTISSWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTF GPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQTISSWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGVPYTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISNWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTF GPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSVGNWLAWYQQKPGKAPKLLIY
GASSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQNIGNWLAWYQQKPGKAPKLLIY
AASTLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYSF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY
GASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSVPYSF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISKWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPYTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSVPYSF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQTISNYLNWYQQKPGKAPKLLIY
AASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPQTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASRDIGRAVGWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLDSYPFTF GPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPYTF GPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTF GGGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSIGRWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYSF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTF AQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY
GASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTF GPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQSVSNWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPTFG QGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYAIPLTF GGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDIGSWLAWYQQKPGKAPKLLIY
ATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPGKAPKLLIY
AASTLQPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYIAPFTF GPGTKVDIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIY
AASRLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTF GGGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY
GVSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPTFG QGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWVAWYQQKPGKAPKLLIY
GASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYSF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSDQWTF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYLPPYSF GQGTKVEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISNWLAWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTYFTLTISSLQPEDFATYYCQQSYSSPYTF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISHYLNWYQQKPGKAPKLLIY
GASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPWTF GQGTRLEIK,
DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYLPPYSF GQGTKLEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY
GASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQEGITYTFGQ GTKVEIK,
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSYPFTF GPGTKVDIK, and
EIVMTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPGQAPRLLIY
GASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYGYSPVTF GQGTKLEIK.
[0437] VH
[0438] The ABP specific for A*01:01_NTDNNLAVY may comprise a VH
sequence. The VH sequence may be selected from
TABLE-US-00031
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSATISWVRQAPGQGLEWMGWIYPNS
GGTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAATEWLGVWGQGTT VTVSS,
EVQLLQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNSG
GTISAPNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARANWLDYWGQGTLVT VSS,
EVQLLESGAEVKKPGASVKVSCKASGYTFTTYDLAWVRQAPGQGLEWMGWINPNS
GGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARANWLDYWGQGT LVTVSS,
QVQLVQSGAEVKKPGASVKVSCKSSGYSFDSYVVNWVRQAPGQGLEWMGWINPN
SGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDWVLDYWGQG TLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWMNPN
SGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGEWLDYWGQGT LVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPNS
GGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGWELGYWGQGTL VTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTINWVRQAPGQGLEWMGWINPNS
GGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDFVGYDDWGQGT LVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGITWVRQAPGQGLEWMGWINPNS
GGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYGDLDYWGQGT LVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSNYILSWVRQAPGQGLEWMGWINPDS
GGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSYGMDVWGQG TTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYSFTRYNMHWVRQAPGQGLEWMGWINPN
SGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGYSGLDVWGK GTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNN
GGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSGVGMDVWGQ GTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFNNYAFSWVRQAPGQGLEWMGWINPN
SGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGVAVASDYWG QGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSSYNMHWVRQAPGQGLEWMGWINGN
TGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVNVDDFDYWG QGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGWINPDT
GYTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDYTGNWYFDLW GRGTLVTVSS,
EVQLLESGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPYSG
GTNYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARANWLDYWGQGTL VTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYN
GYTNYAQNLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDQFYGGNSGGHD
YWGQGTLVTVSS, QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQGLEWMGWMNP
NSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARE- EDYWGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTINWVRQAPGQGLEWMGWINPNS
GGANYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDWFDPWGQGTL VTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYLMHWVRQAPGQGLEWMGWISPN
SGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGDWFDPWGQGT LVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYVHWVRQAPGQGLEWMGWINPN
SGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGEWFDPWGQGT LVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMHWVRQAPGQGLEWMGWINPN
SGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSDWFDPWGQGT LVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSNYAINWVRQAPGQGLEWMGWISPYS
GGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSGSYFDYWGQG TLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWIYPN
TGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDYGGYVDYWG QGTLVTVSS,
EVQLLESGAEVKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWMNPN
SGGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGPAALDVWGQ GTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTLTSHLIHWVRQAPGQGLEWMGWINPNS
GGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARERRSGMDVWGQG TTVTVSS,
EVQLLESGAEVKKPGASVKVSCKASGYSFTDYIVHWVRQAPGQGLEWMGWINPYS
GGTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVLQEGMDVWGQ GTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFSNFLINWVRQAPGQGLEWMGWINPNS
GGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASERELPFDIWGQGT MVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYQMFWVRQAPGQGLEWMGWINPN
SGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKGGGGYGMDVW GQGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNS
GGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAAMGIAVAGGMDV WGQGTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYHMHWVRQAPGQGLEWMGWIHPD
SGGTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNWNLDYWGQGT LVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWMNP
NSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCATYDDGMDVWG QGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTVNWVRQAPGQGLEWMGWINPN
SGGTKYAQNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGGGALDYWGQ GTLVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGMINPR
DDTTDYARDFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCALSGNYYGMDVWG QGTTVTVSS,
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGMINPS
GGGTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGNPWELRLDYW GQGTLVTVSS,
and QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSQYMHWVRQAPGQGLEWMGRIIPLL
GIVNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDKNYYGMDVWGQ
GTTVTVSS.
[0439] VH-VL Combinations
[0440] The ABP specific for A*01:01_NTDNNLAVY may comprise a
particular VH sequence and a particular VL sequence. In some
embodiments, the ABP specific for A*01:01_NTDNNLAVY comprises the
VH sequence and the VL sequence from the scFv designated G2-P2E07,
G2-P2E03, G2-P2A11, G2-P2C06, G2-P1G01, G2-P1C02, G2-P1H01,
G2-P1B12, G2-P1B06, G2-P2H10, G2-P1H10, G2-P2C11, G2-P1C09,
G2-P1A10, G2-P1B10, G2-P1D07, G2-P1E05, G2-P1D03, G2-P1G12,
G2-P2H11, G2-P1C03, G2-P1G07, G2-P1F12, G2-P1G03, G2-P2B08,
G2-P2A10, G2-P2D04, G2-P1C06, G2-P2A09, G2-P1B08, G2-P1E03,
G2-P2A03, G2-P2F01, G2-P1H11, or G2-P1D06. VH and VL sequences of
identified scFvs that specifically bind A*01:01_NTDNNLAVY are found
in Table 33. For clarity, each identified scFv hit is designated a
clone name, and each row contains the CDR sequences for that
particular clone name. For example, the scFv identified by clone
name G2-P2E07 comprises the VH sequence
QVQLVQSGAEVKKPGASVKVSCKASGGTFSSATISWVRQAPGQGLEWMGWIYPNS
GGTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAATEWLGVWGQGTT VTVSSAS and
the VL sequence
TABLE-US-00032 DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYA
ASSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPYTFGQ GTKLEIK.
[0441] Receptors
[0442] Among the provided ABPs, e.g., HLA-PEPTIDE ABPs, are
receptors. The receptors can include antigen receptors and other
chimeric receptors that specifically bind an HLA-PEPTIDE target
disclosed herein. The receptor may be a T cell receptor (TCR). The
receptor may be a chimeric antigen receptor (CAR).
[0443] TCRs can be soluble or membrane-bound. Among the antigen
receptors are functional non-TCR antigen receptors, such as
chimeric antigen receptors (CARs). Also provided are cells
expressing the receptors and uses thereof in adoptive cell therapy,
such as treatment of diseases and disorders associated with
HLA-PEPTIDE expression, including cancer.
[0444] Exemplary antigen receptors, including CARs, and methods for
engineering and introducing such receptors into cells, include
those described, for example, in international patent application
publication numbers WO200014257, WO2013126726, WO2012/129514,
WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S.
patent application publication numbers US2002131960, US2013287748,
US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592,
8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209,
7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent
application number EP2537416, and/or those described by Sadelain et
al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013)
PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012
October; 24(5): 633-39; Wu et al., Cancer, 2012 Mar. 18(2): 160-75.
In some aspects, the antigen receptors include a CAR as described
in U.S. Pat. No. 7,446,190, and those described in International
Patent Application Publication No.: WO/2014055668 A1. Exemplary of
the CARs include CARs as disclosed in any of the aforementioned
publications, such as WO2014031687, U.S. Pat. Nos. 8,339,645,
7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, 8,389,282,
e.g., and in which the antigen-binding portion, e.g., scFv, is
replaced by an antibody, e.g., as provided herein.
[0445] Among the chimeric receptors are chimeric antigen receptors
(CARs). The chimeric receptors, such as CARs, generally include an
extracellular antigen binding domain that includes, is, or is
comprised within, one of the provided anti-HLA-PEPTIDE ABPs such as
anti-HLA-PEPTIDE antibodies. Thus, the chimeric receptors, e.g.,
CARs, typically include in their extracellular portions one or more
HLA-PEPTIDE-ABPs, such as one or more antigen-binding fragment,
domain, or portion, or one or more antibody variable domains,
and/or antibody molecules, such as those described herein. In some
embodiments, the CAR includes a HLA-PEPTIDE-binding portion or
portions of the ABP (e.g., antibody) molecule, such as a variable
heavy (VH) chain region and/or variable light (VL) chain region of
the antibody, e.g., an scFv antibody fragment.
[0446] TCRs
[0447] In an aspect, the ABPs provided herein, e.g., ABPs that
specifically bind HLA-PEPTIDE targets disclosed herein, include T
cell receptors (TCRs). The TCRs may be isolated and purified.
[0448] In a majority of T-cells, the TCR is a heterodimer
polypeptide having an alpha (.alpha.) chain and beta-(.beta.)
chain, encoded by TRA and TRB, respectively. The alpha chain
generally comprises an alpha variable region, encoded by TRAV, an
alpha joining region, encoded by TRAJ, and an alpha constant
region, encoded by TRAC. The beta chain generally comprises a beta
variable region, encoded by TRBV, a beta diversity region, encoded
by TRBD, a beta joining region, encoded by TRBJ, and a beta
constant region, encoded by TRBC. The TCR-alpha chain is generated
by VJ recombination, and the beta chain receptor is generated by
V(D)J recombination. Additional TCR diversity stems from junctional
diversity. Several bases may be deleted and others added (called N
and P nucleotides) at each of the junctions. In a minority of
T-cells, the TCRs include gamma and delta chains. The TCR gamma
chain is generated by VJ recombination, and the TCR delta chain is
generated by V(D)J recombination (Kenneth Murphy, Paul Travers, and
Mark Walport, Janeway's Immunology 7th edition, Garland Science,
2007, which is herein incorporated by reference in its entirety).
The antigen binding site of a TCR generally comprises six
complementarity determining regions (CDRs). The alpha chain
contributes three CDRs, alpha CDR1, alpha CDR2, and .alpha.CDR3.
The beta chain also contributes three CDR: beta CDR1, beta CDR2,
and .beta.CDR3. The .alpha.CDR3 and .beta.CDR3 are the regions most
affected by V(D)J recombination and account for most of the
variation in a TCR repertoire.
[0449] TCRs can specifically recognize HLA-PEPTIDE targets, such as
an HLA-PEPTIDE target disclosed in Table A, A1, or A2; thus TCRs
can be ABPs that specifically bind to HLA-PEPTIDE. TCRs can be
soluble, e.g., similar to an antibody secreted by a B cell. TCRs
can also be membrane-bound, e.g., on a cell such as a T cell or
natural killer (NK) cell. Thus, TCRs can be used in a context that
corresponds to soluble antibodies and/or membrane-bound CARs.
[0450] Any of the TCRs disclosed herein may comprise an alpha
variable region, an alpha joining region, optionally an alpha
constant region, a beta variable region, optionally a beta
diversity region, a beta joining region, and optionally a beta
constant region.
[0451] In some embodiments, the TCR or CAR is a recombinant TCR or
CAR. The recombinant TCR or CAR may include any of the TCRs
identified herein but include one or more modifications. Exemplary
modifications, e.g., amino acid substitutions, are described
herein. Amino acid substitutions described herein may be made with
reference to IMGT nomenclature and amino acid numbering as found at
www.imgt.org.
[0452] The recombinant TCR or CAR may be a human TCR or CAR,
comprising fully human sequences, e.g., natural human sequences.
The recombinant TCR or CAR may retain its natural human variable
domain sequences but contain modifications to the .alpha. constant
region, .beta. constant region, or both .alpha. and .beta. constant
regions. Such modifications to the TCR constant regions may improve
TCR assembly and expression for TCR gene therapy by, e.g., driving
preferential pairings of the exogenous TCR chains.
[0453] In some embodiments, the .alpha. and .beta. constant regions
are modified by substituting the entire human constant region
sequences for mouse constant region sequences. Such "murinized"
TCRs and methods of making them are described in Cancer Res. 2006
Sep. 1; 66(17):8878-86, which is hereby incorporated by reference
in its entirety.
[0454] In some embodiments, the .alpha. and .beta. constant regions
are modified by making one or more amino acid substitutions in the
human TCR .alpha. constant (TRAC) region, the TCR .beta. constant
(TRBC) region, or the TRAC and TRAB regions, which swap particular
human residues for murine residues (human.fwdarw.murine amino acid
exchange). The one or more amino acid substitutions in the TRAC
region may include a Ser substitution at residue 90, an Asp
substitution at residue 91, a Val substitution at residue 92, a Pro
substitution at residue 93, or any combination thereof. The one or
more amino acid substitutions in the human TRBC region may include
a Lys substitution at residue 18, an Ala substitution at residue
22, an Ile substitution at residue 133, a His substitution at
residue 139, or any combination of the above. Such targeted amino
acid substitutions are described in J Immunol Jun. 1, 2010, 184
(11) 6223-6231, which is hereby incorporated by reference in its
entirety.
[0455] In some embodiments, the human TRAC contains an Asp
substitution at residue 210 and the human TRBC contains a Lys
substitution at residue 134. Such substitutions may promote the
formation of a salt bridge between the alpha and beta chains and
formation of the TCR interchain disulfide bond. These targeted
substitutions are described in J Immunol Jun. 1, 2010, 184 (11)
6232-6241, which is hereby incorporated by reference in its
entirety.
[0456] In some embodiments, the human TRAC and human TRBC regions
are modified to contain introduced cysteines which may improve
preferential pairing of the exogenous TCR chains through formation
of an additional disulfide bond. For example, the human TRAC may
contain a Cys substitution at residue 48 and the human TRBC may
contain a Cys substitution at residue 57, described in Cancer Res.
2007 Apr. 15; 67(8):3898-903 and Blood. 2007 Mar. 15;
109(6):2331-8, which are hereby incorporated by reference in their
entirety.
[0457] The recombinant TCR or CAR may comprise other modifications
to the .alpha. and .beta. chains.
[0458] In some embodiments, the .alpha. and .beta. chains are
modified by linking the extracellular domains of the .alpha. and
.beta. chains to a complete human CD3.zeta. (CD3-zeta) molecule.
Such modifications are described in J Immunol Jun. 1, 2008, 180
(11) 7736-7746; Gene Ther. 2000 August; 7(16):1369-77; and The Open
Gene Therapy Journal, 2011, 4: 11-22, which are hereby incorporated
by reference in their entirety.
[0459] In some embodiments, the .alpha. chain is modified by
introducing hydrophobic amino acid substitutions in the
transmembrane region of the .alpha. chain, as described in J
Immunol Jun. 1, 2012, 188 (11) 5538-5546; hereby incorporated by
reference in their entirety.
[0460] The alpha or beta chain may be modified by altering any one
of the N-glycosylation sites in the amino acid sequence, as
described in J Exp Med. 2009 Feb. 16; 206(2): 463-475; hereby
incorporated by reference in its entirety.
[0461] The alpha and beta chain may each comprise a dimerization
domain, e.g., a heterologous dimerization domain. Such a
heterologous domain may be a leucine zipper, a 5H3 domain or
hydrophobic proline rich counter domains, or other similar
modalities, as known in the art. In one example, the alpha and beta
chains may be modified by introducing 30mer segments to the
carboxyl termini of the alpha and beta extracellular domains,
wherein the segments selectively associate to form a stable leucine
zipper. Such modifications are described in PNAS Nov. 22, 1994. 91
(24) 11408-11412; https://doi.org/10.1073/pnas.91.24.11408; hereby
incorporated by reference in its entirety.
[0462] TCRs identified herein may be modified to include mutations
that result in increased affinity or half-life, such as those
described in WO2012/013913, hereby incorporated by reference in its
entirety.
[0463] The recombinant TCR or CAR may be a single chain TCR
(scTCR). Such scTCR may comprise an .alpha. chain variable region
sequence fused to the N terminus of a TCR .alpha. chain constant
region extracellular sequence, a TCR .beta. chain variable region
fused to the N terminus of a TCR .beta. chain constant region
extracellular sequence, and a linker sequence linking the C
terminus of the .alpha. segment to the N terminus of the .beta.
segment, or vice versa. In some embodiments, the constant region
extracellular sequences of the .alpha. and .beta. segments of the
scTCR are linked by a disulfide bond. In some embodiments, the
length of the linker sequence and the position of the disulfide
bond being such that the variable region sequences of the .alpha.
and .beta. segments are mutually orientated substantially as in
native .alpha..beta. T cell receptors. Exemplary scTCRs are
described in U.S. Pat. No. 7,569,664, which is hereby incorporated
by reference in its entirety.
[0464] In some cases, the variable regions of the scTCR may be
covalently joined by a short peptide linker, such as described in
Gene Therapy volume 7, pages 1369-1377 (2000). The short peptide
linker may be a serine rich or glycine rich linker. For example,
the linker may be (Gly.sub.4Ser).sub.3, as described in Cancer Gene
Therapy (2004) 11, 487-496, incorporated by reference in its
entirety.
[0465] The recombinant TCR or antigen binding fragment thereof may
be expressed as a fusion protein. For instance, the TCR or antigen
binding fragment thereof may be fused with a toxin. Such fusion
proteins are described in Cancer Res. 2002 Mar. 15; 62(6):1757-60.
The TCR or antigen binding fragment thereof may be fused with an
antibody Fc region. Such fusion proteins are described in J Immunol
May 1, 2017, 198 (1 Supplement) 120.9.
[0466] In some embodiments, the recombinant receptor such as a TCR
or CAR, such as the antibody portion thereof, further includes a
spacer, which may be or include at least a portion of an
immunoglobulin constant region or variant or modified version
thereof, such as a hinge region, e.g., an IgG4 hinge region, and/or
a CH1/CL and/or Fc region. In some embodiments, the constant region
or portion is of a human IgG, such as IgG4 or IgG1. In some
aspects, the portion of the constant region serves as a spacer
region between the antigen-recognition component, e.g., scFv, and
transmembrane domain. The spacer can be of a length that provides
for increased responsiveness of the cell following antigen binding,
as compared to in the absence of the spacer. In some examples, the
spacer is at or about 12 amino acids in length or is no more than
12 amino acids in length. Exemplary spacers include those having at
least about 10 to 229 amino acids, about 10 to 200 amino acids,
about 10 to 175 amino acids, about 10 to 150 amino acids, about 10
to 125 amino acids, about 10 to 100 amino acids, about 10 to 75
amino acids, about 10 to 50 amino acids, about 10 to 40 amino
acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or
about 10 to 15 amino acids, and including any integer between the
endpoints of any of the listed ranges. In some embodiments, a
spacer region has about 12 amino acids or less, about 119 amino
acids or less, or about 229 amino acids or less. Exemplary spacers
include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains,
or IgG4 hinge linked to the CH3 domain. Exemplary spacers include,
but are not limited to, those described in Hudecek et al. (2013)
Clin. Cancer Res., 19:3153 or international patent application
publication number WO2014031687. In some embodiments, the constant
region or portion is of IgD.
[0467] The antigen recognition domain of a receptor such as a TCR
or CAR can be linked to one or more intracellular signaling
components, such as signaling components that mimic activation
through an antigen receptor complex, such as a TCR complex, in the
case of a CAR, and/or signal via another cell surface receptor.
Thus, in some embodiments, the HLA-PEPTIDE-specific binding
component (e.g., ABP such as antibody or TCR) is linked to one or
more transmembrane and intracellular signaling domains. In some
embodiments, the transmembrane domain is fused to the extracellular
domain. In one embodiment, a transmembrane domain that naturally is
associated with one of the domains in the receptor, e.g., CAR, is
used. In some instances, the transmembrane domain is selected or
modified by amino acid substitution to avoid binding of such
domains to the transmembrane domains of the same or different
surface membrane proteins to minimize interactions with other
members of the receptor complex.
[0468] The transmembrane domain in some embodiments is derived
either from a natural or from a synthetic source. Where the source
is natural, the domain in some aspects is derived from any
membrane-bound or transmembrane protein. Transmembrane regions
include those derived from (i.e. comprise at least the
transmembrane region(s) of) the alpha, beta or zeta chain of the
T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD
16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, and/or CD
154. Alternatively the transmembrane domain in some embodiments is
synthetic. In some aspects, the synthetic transmembrane domain
comprises predominantly hydrophobic residues such as leucine and
valine. In some aspects, a triplet of phenylalanine, tryptophan and
valine will be found at each end of a synthetic transmembrane
domain. In some embodiments, the linkage is by linkers, spacers,
and/or transmembrane domain(s).
[0469] Among the intracellular signaling domains are those that
mimic or approximate a signal through a natural antigen receptor, a
signal through such a receptor in combination with a costimulatory
receptor, and/or a signal through a costimulatory receptor alone.
In some embodiments, a short oligo- or polypeptide linker, for
example, a linker of between 2 and 10 amino acids in length, such
as one containing glycines and serines, e.g., glycine-serine
doublet, is present and forms a linkage between the transmembrane
domain and the cytoplasmic signaling domain of the receptor.
[0470] The receptor, e.g., the TCR or CAR, can include at least one
intracellular signaling component or components. In some
embodiments, the receptor includes an intracellular component of a
TCR complex, such as a TCR CD3 chain that mediates T-cell
activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some
aspects, the HLA-PEPTIDE-binding ABP (e.g., antibody) is linked to
one or more cell signaling modules. In some embodiments, cell
signaling modules include CD3 transmembrane domain, CD3
intracellular signaling domains, and/or other CD transmembrane
domains. In some embodiments, the receptor, e.g., CAR, further
includes a portion of one or more additional molecules such as Fc
receptor-gamma, CD8, CD4, CD25, or CD16. For example, in some
aspects, the CAR includes a chimeric molecule between CD3-zeta or
Fc receptor-gamma and CD8, CD4, CD25 or CD16.
[0471] In some embodiments, upon ligation of the TCR or CAR, the
cytoplasmic domain or intracellular signaling domain of the
receptor activates at least one of the normal effector functions or
responses of the immune cell, e.g., T cell engineered to express
the receptor. For example, in some contexts, the receptor induces a
function of a T cell such as cytolytic activity or T-helper
activity, such as secretion of cytokines or other factors. In some
embodiments, a truncated portion of an intracellular signaling
domain of an antigen receptor component or costimulatory molecule
is used in place of an intact immunostimulatory chain, for example,
if it transduces the effector function signal. In some embodiments,
the intracellular signaling domain or domains include the
cytoplasmic sequences of the T cell receptor (TCR), and in some
aspects also those of co-receptors that in the natural context act
in concert with such receptor to initiate signal transduction
following antigen receptor engagement, and/or any derivative or
variant of such molecules, and/or any synthetic sequence that has
the same functional capability.
[0472] In the context of a natural TCR, full activation generally
uses not only signaling through the TCR, but also a costimulatory
signal. Thus, in some embodiments, to promote full activation, a
component for generating secondary or co-stimulatory signal is also
included in the receptor. In other embodiments, the receptor does
not include a component for generating a costimulatory signal. In
some aspects, an additional receptor is expressed in the same cell
and provides the component for generating the secondary or
costimulatory signal.
[0473] T cell activation is in some aspects described as being
mediated by two classes of cytoplasmic signaling sequences: those
that initiate antigen-dependent primary activation through the TCR
(primary cytoplasmic signaling sequences), and those that act in an
antigen-independent manner to provide a secondary or co-stimulatory
signal (secondary cytoplasmic signaling sequences). In some
aspects, the receptor includes one or both of such signaling
components.
[0474] In some aspects, the receptor includes a primary cytoplasmic
signaling sequence that regulates primary activation of the TCR
complex. Primary cytoplasmic signaling sequences that act in a
stimulatory manner may contain signaling motifs which are known as
immunoreceptor tyrosine-based activation motifs or ITAMs. Examples
of ITAM containing primary cytoplasmic signaling sequences include
those derived from TCR or CD3 zeta, FcR gamma, FcR beta, CD3 gamma,
CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some
embodiments, cytoplasmic signaling molecule(s) in the CAR
contain(s) a cytoplasmic signaling domain, portion thereof, or
sequence derived from CD3 zeta.
[0475] In some embodiments, the receptor includes a signaling
domain and/or transmembrane portion of a costimulatory receptor,
such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the
same receptor includes both the activating and costimulatory
components.
[0476] In some embodiments, the activating domain is included
within one receptor, whereas the costimulatory component is
provided by another receptor recognizing another antigen. In some
embodiments, the receptors include activating or stimulatory
receptors, and costimulatory receptors, both expressed on the same
cell (see WO2014/055668). In some aspects, the
HLA-PEPTIDE-targeting receptor is the stimulatory or activating
receptor; in other aspects, it is the costimulatory receptor. In
some embodiments, the cells further include inhibitory receptors
(e.g., iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215)
(December, 2013), such as a receptor recognizing an antigen other
than HLA-PEPTIDE, whereby an activating signal delivered through
the HLA-PEPTIDE-targeting receptor is diminished or inhibited by
binding of the inhibitory receptor to its ligand, e.g., to reduce
off-target effects.
[0477] In certain embodiments, the intracellular signaling domain
comprises a CD28 transmembrane and signaling domain linked to a CD3
(e.g., CD3-zeta) intracellular domain. In some embodiments, the
intracellular signaling domain comprises a chimeric CD28 and CD137
(4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta
intracellular domain.
[0478] In some embodiments, the receptor encompasses one or more,
e.g., two or more, costimulatory domains and an activation domain,
e.g., primary activation domain, in the cytoplasmic portion.
Exemplary receptors include intracellular components of CD3-zeta,
CD28, and 4-1BB.
[0479] In some embodiments, the CAR or other antigen receptor such
as a TCR further includes a marker, such as a cell surface marker,
which may be used to confirm transduction or engineering of the
cell to express the receptor, such as a truncated version of a cell
surface receptor, such as truncated EGFR (tEGFR). In some aspects,
the marker includes all or part (e.g., truncated form) of CD34, a
nerve growth factor receptor (NGFR), or epidermal growth factor
receptor (e.g., tEGFR). In some embodiments, the nucleic acid
encoding the marker is operably linked to a polynucleotide encoding
for a linker sequence, such as a cleavable linker sequence or a
ribosomal skip sequence, e.g., T2A. See WO2014031687. In some
embodiments, introduction of a construct encoding the CAR and EGFRt
separated by a T2A ribosome switch can express two proteins from
the same construct, such that the EGFRt can be used as a marker to
detect cells expressing such construct. In some embodiments, a
marker, and optionally a linker sequence, can be any as disclosed
in published patent application No. WO2014031687. For example, the
marker can be a truncated EGFR (tEGFR) that is, optionally, linked
to a linker sequence, such as a T2A ribosomal skip sequence.
[0480] In some embodiments, the marker is a molecule, e.g., cell
surface protein, not naturally found on T cells or not naturally
found on the surface of T cells, or a portion thereof.
[0481] In some embodiments, the molecule is a non-self molecule,
e.g., non-self protein, i.e., one that is not recognized as "self"
by the immune system of the host into which the cells will be
adoptively transferred.
[0482] In some embodiments, the marker serves no therapeutic
function and/or produces no effect other than to be used as a
marker for genetic engineering, e.g., for selecting cells
successfully engineered. In other embodiments, the marker may be a
therapeutic molecule or molecule otherwise exerting some desired
effect, such as a ligand for a cell to be encountered in vivo, such
as a costimulatory or immune checkpoint molecule to enhance and/or
dampen responses of the cells upon adoptive transfer and encounter
with ligand.
[0483] The TCR or CAR may comprise one or modified synthetic amino
acids in place of one or more naturally-occurring amino acids.
Exemplary modified amino acids include, but are not limited to,
aminocyclohexane carboxylic acid, norleucine, .alpha.-amino
n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3-
and trans-4-hydroxyproline, 4-aminophenylalanine,
4-nitrophenylalanine, 4-chlorophenylalanine,
4-carboxyphenylalanine, (3-phenylserine (3-hydroxyphenylalanine,
phenylglycine, .alpha.-naphthylalanine, cyclohexylalanine,
cyclohexylglycine, indoline-2-carboxylic acid,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic
acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine,
N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine,
.alpha.-aminocyclopentane carboxylic acid, .alpha.-aminocyclohexane
carboxylic acid, .alpha.-aminocycloheptane carboxylic acid,
.alpha.-(2-amino-2-norbornane)-carboxylic acid,
.alpha.,.gamma.-diaminobutyric acid,
.alpha.,.gamma.-diaminopropionic acid, homophenylalanine, and
.alpha.-tertbutylglycine.
[0484] In some cases, CARs are referred to as first, second, and/or
third generation CARs. In some aspects, a first generation CAR is
one that solely provides a CD3-chain induced signal upon antigen
binding; in some aspects, a second-generation CARs is one that
provides such a signal and costimulatory signal, such as one
including an intracellular signaling domain from a costimulatory
receptor such as CD28 or CD137; in some aspects, a third generation
CAR in some aspects is one that includes multiple costimulatory
domains of different costimulatory receptors.
[0485] In some embodiments, the chimeric antigen receptor includes
an extracellular portion containing an antibody or fragment
described herein. In some aspects, the chimeric antigen receptor
includes an extracellular portion containing an antibody or
fragment described herein and an intracellular signaling domain. In
some embodiments, an antibody or fragment includes an scFv or a
single-domain VH antibody and the intracellular domain contains an
ITAM. In some aspects, the intracellular signaling domain includes
a signaling domain of a zeta chain of a CD3-zeta (CD3) chain. In
some embodiments, the chimeric antigen receptor includes a
transmembrane domain linking the extracellular domain and the
intracellular signaling domain.
[0486] In some aspects, the transmembrane domain contains a
transmembrane portion of CD28. The extracellular domain and
transmembrane can be linked directly or indirectly. In some
embodiments, the extracellular domain and transmembrane are linked
by a spacer, such as any described herein. In some embodiments, the
chimeric antigen receptor contains an intracellular domain of a T
cell costimulatory molecule, such as between the transmembrane
domain and intracellular signaling domain. In some aspects, the T
cell costimulatory molecule is CD28 or 41BB.
[0487] In some embodiments, the CAR contains an antibody, e.g., an
antibody fragment, a transmembrane domain that is or contains a
transmembrane portion of CD28 or a functional variant thereof, and
an intracellular signaling domain containing a signaling portion of
CD28 or functional variant thereof and a signaling portion of CD3
zeta or functional variant thereof. In some embodiments, the CAR
contains an antibody, e.g., antibody fragment, a transmembrane
domain that is or contains a transmembrane portion of CD28 or a
functional variant thereof, and an intracellular signaling domain
containing a signaling portion of a 4-1BB or functional variant
thereof and a signaling portion of CD3 zeta or functional variant
thereof. In some such embodiments, the receptor further includes a
spacer containing a portion of an Ig molecule, such as a human Ig
molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a
hinge-only spacer.
[0488] In some embodiments, the transmembrane domain of the
receptor, e.g., the CAR, is a transmembrane domain of human CD28 or
variant thereof, e.g., a 27-amino acid transmembrane domain of a
human CD28 (Accession No.: P10747.1).
[0489] In some embodiments, the chimeric antigen receptor contains
an intracellular domain of a T cell costimulatory molecule. In some
aspects, the T cell costimulatory molecule is CD28 or 41BB.
[0490] In some embodiments, the intracellular signaling domain
comprises an intracellular costimulatory signaling domain of human
CD28 or functional variant or portion thereof, such as a 41 amino
acid domain thereof and/or such a domain with an LL to GG
substitution at positions 186-187 of a native CD28 protein. In some
embodiments, the intracellular domain comprises an intracellular
costimulatory signaling domain of 41BB or functional variant or
portion thereof, such as a 42-amino acid cytoplasmic domain of a
human 4-1BB (Accession No. Q07011.1) or functional variant or
portion thereof.
[0491] In some embodiments, the intracellular signaling domain
comprises a human CD3 zeta stimulatory signaling domain or
functional variant thereof, such as a 112 AA cytoplasmic domain of
isoform 3 of human CD3.zeta. (Accession No.: P20963.2) or a CD3
zeta signaling domain as described in U.S. Pat. No. 7,446,190 or
8,911,993.
[0492] In some aspects, the spacer contains only a hinge region of
an IgG, such as only a hinge of IgG4 or IgG1. In other embodiments,
the spacer is an Ig hinge, e.g., and IgG4 hinge, linked to a CH2
and/or CH3 domains. In some embodiments, the spacer is an Ig hinge,
e.g., an IgG4 hinge, linked to CH2 and CH3 domains. In some
embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked
to a CH3 domain only. In some embodiments, the spacer is or
comprises a glycine-serine rich sequence or other flexible linker
such as known flexible linkers.
[0493] For example, in some embodiments, the CAR includes an
antibody or fragment thereof, such as any of the HLA-PEPTIDE
antibodies, including single chain antibodies (sdAbs, e.g.
containing only the VH region) and scFvs, described herein, a
spacer such as any of the Ig-hinge containing spacers, a CD28
transmembrane domain, a CD28 intracellular signaling domain, and a
CD3 zeta signaling domain. In some embodiments, the CAR includes an
antibody or fragment, such as any of the HLA-PEPTIDE antibodies,
including sdAbs and scFvs described herein, a spacer such as any of
the Ig-hinge containing spacers, a CD28 transmembrane domain, a
CD28 intracellular signaling domain, and a CD3 zeta signaling
domain.
Target-Specific TCRs to A*01:01 ASSLPTTMNY [G10]
[0494] In some aspects, provided herein are ABPs comprising TCRs or
antigen-binding fragments thereof that specifically bind an
HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype A*01:01 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises the sequence ASSLPTTMNY
("G10").
[0495] The TCR specific for A*01:01_ASSLPTTMNY may comprise an
.alpha.CDR3 sequence. The .alpha.CDR3 sequence may be any one of
the .alpha.CDR3 sequences in Table 15. Refer to PCT/US2018/06793,
filed on Dec. 28, 2018, which is hereby incorporated by reference
in its entirety. Alpha and beta CDR3 sequences of the identified
TCR clonotypes are shown in Table 15.
[0496] The TCR specific for A*01:01_ASSLPTTMNY may comprise a
.beta.CDR3 sequence. The .beta.CDR3 sequence may be any one of the
.beta.CDR3 sequences in Table 15. Refer to PCT/US2018/06793, filed
on Dec. 28, 2018, which is hereby incorporated by reference in its
entirety.
[0497] The TCR specific for A*01:01_ASSLPTTMNY may comprise a
particular .alpha.CDR3 sequence and a particular .beta.CDR3
sequence. For example, the TCR specific for A*01:01_ASSLPTTMNY may
comprise the .alpha.CDR3 sequence and .beta.CDR3 sequence from any
one of TCRs identified in Table 15. Refer to PCT/US2018/06793,
filed on Dec. 28, 2018, which is hereby incorporated by reference
in its entirety.
[0498] The TCR specific for A*01:01_ASSLPTTMNY may comprise a TRAV,
a TRAJ, a TRBV, optionally a TRBD, and a TRBJ amino acid sequence,
optionally a TRAC sequence and optionally a TRBC sequence. For
example, the TCR specific for A*01:01_ASSLPTTMNY may comprise the
TRAV, TRAJ, TRBV, TRBD, TRBJ amino acid sequence, TRAC sequence and
TRBC sequence from any one of the TCRs identified in Table 14.
Refer to PCT/US2018/06793, filed on Dec. 28, 2018, which is hereby
incorporated by reference in its entirety. For clarity, each
identified TCR was assigned a TCR ID number. For example the TCR
assigned TCR ID #1 comprises a TRAV25 sequence, a TRAJ37 sequence,
a TRAC sequence, a TRBV19 sequence, a TRBD1 sequence, a TRBJ1-5
sequence, and a TRBC1 sequence.
[0499] The TCR specific for A*01:01_ASSLPTTMNY may comprise an
alpha VJ sequence. The alpha VJ sequence may be any one of the
alpha VJ sequences in Table 16. Refer to PCT/US2018/06793, filed on
Dec. 28, 2018, which is hereby incorporated by reference in its
entirety.
[0500] The TCR specific for A*01:01_ASSLPTTMNY may comprise a beta
V(D)J sequence. The beta V(D)J sequence may be any one of the beta
V(D)J sequences in Table 16. Refer to PCT/US2018/06793, filed on
Dec. 28, 2018, which is hereby incorporated by reference in its
entirety.
[0501] The TCR specific for A*01:01_ASSLPTTMNY may comprise an
alpha VJ sequence and a beta V(D)J sequence. For example, the TCR
specific for A*01:01_ASSLPTTMNY may comprise the alpha VJ sequence
and the beta V(D)J sequence from any one of the TCRs identified in
Table 16. Refer to PCT/US2018/06793, filed on Dec. 28, 2018, which
is hereby incorporated by reference in its entirety. Full length
alpha V(J) and beta V(D)J sequences of the identified TCR
clonotypes are shown in Table 16. Refer to PCT/US2018/06793, filed
on Dec. 28, 2018, which is hereby incorporated by reference in its
entirety.
[0502] Target-Specific TCRs to A*01:01 HSEVGLPVY
[0503] In some aspects, provided herein are ABPs comprising TCRs or
antigen-binding fragments thereof that specifically bind an
HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype A*01:01 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises the sequence
HSEVGLPVY
[0504] The TCR specific for A*01:01_HSEVGLPVY may comprise an
.alpha.CDR3 sequence. The .alpha.CDR3 sequence may be any one of
the .alpha.CDR3 sequences in Table 18. Refer to PCT/US2018/06793,
filed on Dec. 28, 2018, which is hereby incorporated by reference
in its entirety. Alpha and beta CDR3 sequences of the identified
TCR clonotypes are shown in Table 18.
[0505] The TCR specific for A*01:01_HSEVGLPVY may comprise a
.beta.CDR3 sequence. The .beta.CDR3 sequence may be any one of the
.beta.CDR3 sequences in Table 18. Refer to PCT/US2018/06793, filed
on Dec. 28, 2018, which is hereby incorporated by reference in its
entirety.
[0506] The TCR specific for A*01:01_HSEVGLPVY may comprise a
particular .alpha.CDR3 sequence and a particular .beta.CDR3
sequence. For example, the TCR specific for A*01:01_HSEVGLPVY may
comprise the .alpha.CDR3 sequence and .beta.CDR3 sequence from any
one of TCRs identified in Table 18. Refer to PCT/US2018/06793,
filed on Dec. 28, 2018, which is hereby incorporated by reference
in its entirety.
[0507] The TCR specific for A*01:01_HSEVGLPVY may comprise a TRAV,
a TRAJ, a TRBV, optionally a TRBD, and a TRBJ amino acid sequence,
optionally a TRAC sequence and optionally a TRBC sequence. For
example, the TCR specific for A*01:01_HSEVGLPVY may comprise the
TRAV, TRAJ, TRBV, TRBD, TRBJ amino acid sequence, TRAC sequence and
TRBC sequence from any one of the TCRs identified in Table 17.
Refer to PCT/US2018/06793, filed on Dec. 28, 2018, which is hereby
incorporated by reference in its entirety.
[0508] The TCR specific for A*01:01_HSEVGLPVY may comprise an alpha
VJ sequence. The alpha VJ sequence may be any one of the alpha VJ
sequences in Table 19. Refer to PCT/US2018/06793, filed on Dec. 28,
2018, which is hereby incorporated by reference in its
entirety.
[0509] The TCR specific for A*01:01_HSEVGLPVY may comprise a beta
V(D)J sequence. The beta V(D)J sequence may be any one of the beta
V(D)J sequences in Table 19. Refer to PCT/US2018/06793, filed on
Dec. 28, 2018, which is hereby incorporated by reference in its
entirety.
[0510] The TCR specific for A*01:01_HSEVGLPVY may comprise an alpha
VJ sequence and a beta V(D)J sequence. For example, the TCR
specific for A*01:01_HSEVGLPVY may comprise the alpha VJ sequence
and the beta V(D)J sequence from any one of the TCRs identified in
Table 19. Refer to PCT/US2018/06793, filed on Dec. 28, 2018, which
is hereby incorporated by reference in its entirety.
[0511] Target-Specific TCRs to A*02:01 LLASSILCA [G7]
[0512] In some aspects, provided herein are ABPs comprising TCRs or
antigen-binding fragments thereof that specifically bind an
HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype A*02:01 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises the sequence
LLASSILCA.
[0513] The TCR specific for A*02:01__LLASSILCA may comprise an
.alpha.CDR3 sequence. Refer to SEQ ID NO: 4277, 4278, 4279, 4280,
or 4281 of PCT/US2018/046997, filed on Aug. 17, 2018, which
application is incorporated by reference in its entirety.
[0514] The TCR specific for A*02:01__LLASSILCA may comprise a
.beta.CDR3 sequence. Refer to SEQ ID NOS 4291-4295 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0515] The TCR specific for A*02:01__LLASSILCA may comprise a
particular .alpha.CDR3 sequence and a particular .beta.CDR3
sequence. For particular combinations of .alpha.CDR3 and .beta.CDR3
sequences, refer to PCT/US2018/046997, filed on Aug. 17, 2018,
which application is incorporated by reference in its entirety.
[0516] The TCR specific for A*02:01__LLASSILCA may comprise a TRAV,
a TRAJ, a TRBV, optionally a TRBD, and a TRBJ amino acid sequence,
optionally a TRAC sequence and optionally a TRBC sequence. For
particular combinations of TRAV, TRAJ, TRBV, optionally TRBD, TRBJ
amino acid sequence, optionally TRAC sequence and optionally TRBC
sequences, refer to PCT/US2018/046997, filed on Aug. 17, 2018,
which application is incorporated by reference in its entirety.
[0517] The TCR specific for A*02:01__LLASSILCA may comprise an
alpha VJ sequence. Refer to SEQ ID NOS 4306-4310 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0518] The TCR specific for A*02:01__LLASSILCA may comprise a beta
V(D)J sequence. Refer to SEQ ID NOS 4321-4325 of PCT/US2018/046997,
filed on Aug. 17, 2018, which application is incorporated by
reference in its entirety. The TCR specific for A*02:01__LLASSILCA
may comprise an alpha VJ sequence and a beta V(D)J sequence. For
particular combinations of alpha VJ and beta V(D)J sequences, refer
to PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0519] Target-Specific TCRs to A*01:01 EVDPIGHLY
[0520] In some aspects, provided herein are ABPs comprising TCRs or
antigen-binding fragments thereof that specifically bind an
HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype A*01:01 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises the sequence
EVDPIGHLY.
[0521] The TCR specific for A*01:01_EVDPIGHLY may comprise an
.alpha.CDR3 sequence. Refer to SEQ ID NOS 3052-3350 or 4273-4276 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0522] The TCR specific for A*01:01_EVDPIGHLY may comprise a
.beta.CDR3 sequence. Refer to SEQ ID NOS 3351-3655 or 4287-4290 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety
[0523] The TCR specific for A*01:01_EVDPIGHLY may comprise a
particular .alpha.CDR3 sequence and a particular .beta.CDR3
sequence. For particular combinations of .alpha.CDR3 and .beta.CDR3
sequences, refer to PCT/US2018/046997, filed on Aug. 17, 2018,
which application is incorporated by reference in its entirety.
[0524] The TCR specific for A*01:01_EVDPIGHLY may comprise a TRAV,
a TRAJ, a TRBV, optionally a TRBD, and a TRBJ amino acid sequence,
optionally a TRAC sequence and optionally a TRBC sequence. For
particular combinations of TRAV, TRAJ, TRBV, optionally TRBD, TRBJ
amino acid sequence, optionally TRAC sequence and optionally TRBC
sequences, refer to PCT/US2018/046997, filed on Aug. 17, 2018,
which application is incorporated by reference in its entirety.
[0525] The TCR specific for A*01:01_EVDPIGHLY may comprise an alpha
VJ sequence. Refer to SEQ ID NOS 3656-3961 or 4302-4305 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0526] The TCR specific for A*01:01_EVDPIGHLY may comprise a beta
V(D)J sequence. Refer to SEQ ID NOS 3962-4269 or 4317-4320 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0527] The TCR specific for A*01:01_EVDPIGHLY may comprise an alpha
VJ sequence and a beta V(D)J sequence. For particular combinations
of alpha VJ and beta V(D)J sequences, refer to PCT/US2018/046997,
filed on Aug. 17, 2018, which application is incorporated by
reference in its entirety.
Target-Specific TCRs to B*44:02 GEMSSNSTAL
[0528] In some aspects, provided herein are ABPs comprising TCRs or
antigen-binding fragments thereof that specifically bind an
HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype B*44:02 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises the sequence
GEMSSNSTAL.
[0529] The TCR specific for B*44:02_GEMSSNSTAL may comprise an
.alpha.CDR3 sequence. Refer to SEQ ID NOS 4284-4286 or 3138 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0530] The TCR specific for B*44:02_GEMSSNSTAL may comprise a
.beta.CDR3 sequence. Refer to SEQ ID NOS 4298-4301 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0531] The TCR specific for B*44:02_GEMSSNSTAL may comprise a
particular .alpha.CDR3 sequence and a particular .beta.CDR3
sequence. For particular combinations of .alpha.CDR3 and .beta.CDR3
sequences, refer to PCT/US2018/046997, filed on Aug. 17, 2018,
which application is incorporated by reference in its entirety.
[0532] The TCR specific for B*44:02_GEMSSNSTAL may comprise a TRAV,
a TRAJ, a TRBV, optionally a TRBD, and a TRBJ amino acid sequence,
optionally a TRAC sequence and optionally a TRBC sequence. For
particular combinations of TRAV, TRAJ, TRBV, optionally TRBD, TRBJ
amino acid sequence, optionally TRAC sequence and optionally TRBC
sequences, refer to PCT/US2018/046997, filed on Aug. 17, 2018,
which application is incorporated by reference in its entirety.
[0533] The TCR specific for B*44:02_GEMSSNSTAL may comprise an
alpha VJ sequence. Refer to SEQ ID NOS 4313-4316 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0534] The TCR specific for B*44:02_GEMSSNSTAL may comprise a beta
V(D)J sequence. Refer to SEQ ID NOS 4328-4331 of PCT/US2018/046997,
filed on Aug. 17, 2018, which application is incorporated by
reference in its entirety.
[0535] The TCR specific for B*44:02_GEMSSNSTAL may comprise an
alpha VJ sequence and a beta V(D)J sequence. For particular
combinations of alpha VJ and beta V(D)J sequences, refer to
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
Target-Specific TCRs to A*02:01 GVYDGEEHSV
[0536] In some aspects, provided herein are ABPs comprising TCRs or
antigen-binding fragments thereof that specifically bind an
HLA-PEPTIDE target, wherein the HLA Class I molecule of the
HLA-PEPTIDE target is HLA subtype A*02:01 and the HLA-restricted
peptide of the HLA-PEPTIDE target comprises the sequence
GVYDGEEHSV.
[0537] The TCR specific for A*02:01_GVYDGEEHSV may comprise an
.alpha.CDR3 sequence. Refer to SEQ ID NOS 4282-4283 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0538] The TCR specific for A*02:01 GVYDGEEHSV may comprise a
.beta.CDR3 sequence. Refer to SEQ ID NOS 4296-4297 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0539] The TCR specific for A*02:01_GVYDGEEHSV may comprise a
particular .alpha.CDR3 sequence and a particular .beta.CDR3
sequence. For particular combinations of .alpha.CDR3 and .beta.CDR3
sequences, refer to PCT/US2018/046997, filed on Aug. 17, 2018,
which application is incorporated by reference in its entirety.
[0540] The TCR specific for A*02:01_GVYDGEEHSV may comprise a TRAV,
a TRAJ, a TRBV, optionally a TRBD, and a TRBJ amino acid sequence,
optionally a TRAC sequence and optionally a TRBC sequence. For
particular combinations of TRAV, TRAJ, TRBV, optionally TRBD, TRBJ
amino acid sequence, optionally TRAC sequence and optionally TRBC
sequences, refer to PCT/US2018/046997, filed on Aug. 17, 2018,
which application is incorporated by reference in its entirety.
[0541] The TCR specific for A*02:01_GVYDGEEHSV may comprise an
alpha VJ sequence. Refer to SEQ ID NOS 4311-4312 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[0542] The TCR specific for A*02:01_GVYDGEEHSV may comprise a beta
V(D)J sequence. Refer to SEQ ID NOS 4326-4327 of PCT/US2018/046997,
filed on Aug. 17, 2018, which application is incorporated by
reference in its entirety.
[0543] The TCR specific for A*02:01_GVYDGEEHSV may comprise an
alpha VJ sequence and a beta V(D)J sequence. For particular
combinations of alpha VJ and beta V(D)J sequences, refer to
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
Engineered Cells
[0544] Also provided are cells such as cells that contain an
antigen receptor, e.g., that contains an extracellular domain
including an anti-HLA-PEPTIDE ABP (e.g., a CAR or TCR), described
herein. Also provided are populations of such cells, and
compositions containing such cells. In some embodiments,
compositions or populations are enriched for such cells, such as in
which cells expressing the HLA-PEPTIDE ABP make up at least 1, 5,
10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, or more than 99 percent of the total cells in the composition
or cells of a certain type such as T cells or CD8+ or CD4+ cells.
In some embodiments, a composition comprises at least one cell
containing an antigen receptor disclosed herein. Among the
compositions are pharmaceutical compositions and formulations for
administration, such as for adoptive cell therapy. Also provided
are therapeutic methods for administering the cells and
compositions to subjects, e.g., patients.
[0545] Thus also provided are genetically engineered cells
expressing an ABP comprising a receptor, e.g., a TCR or CAR. The
cells generally are eukaryotic cells, such as mammalian cells, and
typically are human cells. In some embodiments, the cells are
derived from the blood, bone marrow, lymph, or lymphoid organs, are
cells of the immune system, such as cells of the innate or adaptive
immunity, e.g., myeloid or lymphoid cells, including lymphocytes,
typically T cells and/or NK cells. Other exemplary cells include
stem cells, such as multipotent and pluripotent stem cells,
including induced pluripotent stem cells (iPSCs). The cells
typically are primary cells, such as those isolated directly from a
subject and/or isolated from a subject and frozen. In some
embodiments, the cells include one or more subsets of T cells or
other cell types, such as whole T cell populations, CD4+ cells,
CD8+ cells, and subpopulations thereof, such as those defined by
function, activation state, maturity, potential for
differentiation, expansion, recirculation, localization, and/or
persistence capacities, antigen-specificity, type of antigen
receptor, presence in a particular organ or compartment, marker or
cytokine secretion profile, and/or degree of differentiation. With
reference to the subject to be treated, the cells may be allogeneic
and/or autologous. Among the methods include off-the-shelf methods.
In some aspects, such as for off-the-shelf technologies, the cells
are pluripotent and/or multipotent, such as stem cells, such as
induced pluripotent stem cells (iPSCs). In some embodiments, the
methods include isolating cells from the subject, preparing,
processing, culturing, and/or engineering them, as described
herein, and re-introducing them into the same patient, before or
after cryopreservation.
[0546] Among the sub-types and subpopulations of T cells and/or of
CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T
cells (TEFF), memory T cells and sub-types thereof, such as stem
cell memory T (TSCM), central memory T (TCM), effector memory T
(TEM), or terminally differentiated effector memory T cells,
tumor-infiltrating lymphocytes (TIL), immature T cells, mature T
cells, helper T cells, cytotoxic T cells, mucosa-associated
invariant T (MALT) cells, naturally occurring and adaptive
regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2
cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular
helper T cells, alpha/beta T cells, and delta/gamma T cells.
[0547] In some embodiments, the cells are natural killer (NK)
cells. In some embodiments, the cells are monocytes or
granulocytes, e.g., myeloid cells, macrophages, neutrophils,
dendritic cells, mast cells, eosinophils, and/or basophils.
[0548] The cells may be genetically modified to reduce expression
or knock out endogenous TCRs. Such modifications are described in
Mol Ther Nucleic Acids. 2012 December; 1(12): e63; Blood. 2011 Aug.
11; 118(6):1495-503; Blood. 2012 Jun. 14; 119(24): 5697-5705;
Torikai, Hiroki et al "HLA and TCR Knockout by Zinc Finger
Nucleases: Toward "off-the-Shelf" Allogeneic T-Cell Therapy for
CD19+ Malignancies." Blood 116.21 (2010): 3766; Blood. 2018 Jan.
18; 131(3):311-322. doi: 10.1182/blood-2017-05-787598; and
WO2016069283, which are incorporated by reference in their
entirety.
[0549] The cells may be genetically modified to promote cytokine
secretion. Such modifications are described in Hsu C, Hughes M S,
Zheng Z, Bray R B, Rosenberg S A, Morgan R A. Primary human T
lymphocytes engineered with a codon-optimized IL-15 gene resist
cytokine withdrawal-induced apoptosis and persist long-term in the
absence of exogenous cytokine. J Immunol. 2005; 175:7226-34;
Quintarelli C, Vera J F, Savoldo B, Giordano Attianese G M, Pule M,
Foster A E, Co-expression of cytokine and suicide genes to enhance
the activity and safety of tumor-specific cytotoxic T lymphocytes.
Blood. 2007; 110:2793-802; and Hsu C, Jones S A, Cohen C J, Zheng
Z, Kerstann K, Zhou J, Cytokine-independent growth and clonal
expansion of a primary human CD8+ T-cell clone following retroviral
transduction with the IL-15 gene. Blood. 2007; 109:5168-77.
[0550] Mismatching of chemokine receptors on T cells and
tumor-secreted chemokines has been shown to account for the
suboptimal trafficking of T cells into the tumor microenvironment.
To improve efficacy of therapy, the cells may be genetically
modified to increase recognition of chemokines in tumor micro
environment. Examples of such modifications are described in Moon
et al., Expression of a functional CCR2 receptor enhances tumor
localization and tumor eradication by retargeted human T cells
expressing a mesothelin-specific chimeric antibody receptor, Clin
Cancer Res. 2011; 17: 4719-4730; and Craddock et al., Enhanced
tumor trafficking of GD2 chimeric antigen receptor T cells by
expression of the chemokine receptor CCR2b. J Immunother. 2010; 33:
780-788.
[0551] The cells may be genetically modified to enhance expression
of costimulatory/enhancing receptors, such as CD28 and 41BB.
[0552] Adverse effects of T cell therapy can include cytokine
release syndrome and prolonged B-cell depletion. Introduction of a
suicide/safety switch in the recipient cells may improve the safety
profile of a cell-based therapy. Accordingly, the cells may be
genetically modified to include a suicide/safety switch. The
suicide/safety switch may be a gene that confers sensitivity to an
agent, e.g., a drug, upon the cell in which the gene is expressed,
and which causes the cell to die when the cell is contacted with or
exposed to the agent. Exemplary suicide/safety switches are
described in Protein Cell. 2017 August; 8(8): 573-589. The
suicide/safety switch may be HSV-TK. The suicide/safety switch may
be cytosine deaminase, purine nucleoside phosphorylase, or
nitroreductase. The suicide/safety switch may be RapaCIDe.TM.,
described in U.S. Patent Application Pub. No. US20170166877A1. The
suicide/safety switch system may be CD20/Rituximab, described in
Haematologica. 2009 September; 94(9): 1316-1320. These references
are incorporated by reference in their entirety.
[0553] The TCR or CAR may be introduced into the recipient cell as
a split receptor which assembles only in the presence of a
heterodimerizing small molecule. Such systems are described in
Science. 2015 Oct. 16; 350(6258): aab4077, and in U.S. Pat. No.
9,587,020, which are hereby incorporated by reference.
[0554] In some embodiments, the cells include one or more nucleic
acids, e.g., a polynucleotide encoding a TCR or CAR disclosed
herein, wherein the polynucleotide is introduced via genetic
engineering, and thereby express recombinant or genetically
engineered TCRs or CARs as disclosed herein. In some embodiments,
the nucleic acids are heterologous, i.e., normally not present in a
cell or sample obtained from the cell, such as one obtained from
another organism or cell, which for example, is not ordinarily
found in the cell being engineered and/or an organism from which
such cell is derived. In some embodiments, the nucleic acids are
not naturally occurring, such as a nucleic acid not found in
nature, including one comprising chimeric combinations of nucleic
acids encoding various domains from multiple different cell
types.
[0555] The nucleic acids may include a codon-optimized nucleotide
sequence. Without being bound to a particular theory or mechanism,
it is believed that codon optimization of the nucleotide sequence
increases the translation efficiency of the mRNA transcripts. Codon
optimization of the nucleotide sequence may involve substituting a
native codon for another codon that encodes the same amino acid,
but can be translated by tRNA that is more readily available within
a cell, thus increasing translation efficiency. Optimization of the
nucleotide sequence may also reduce secondary mRNA structures that
would interfere with translation, thus increasing translation
efficiency.
[0556] A construct or vector may be used to introduce the TCR or
CAR into the recipient cell. Exemplary constructs are described
herein. Polynucleotides encoding the alpha and beta chains of the
TCR or CAR may in a single construct or in separate constructs. The
polynucleotides encoding the alpha and beta chains may be operably
linked to a promoter, e.g., a heterologous promoter. The
heterologous promoter may be a strong promoter, e.g., EF1alpha,
CMV, PGK1, Ubc, beta actin, CAG promoter, and the like. The
heterologous promoter may be a weak promoter. The heterologous
promoter may be an inducible promoter. Exemplary inducible
promoters include, but are not limited to TRE, NFAT, GAL4, LAC, and
the like. Other exemplary inducible expression systems are
described in U.S. Pat. Nos. 5,514,578; 6,245,531; 7,091,038 and
European Patent No. 0517805, which are incorporated by reference in
their entirety.
[0557] The construct for introducing the TCR or CAR into the
recipient cell may also comprise a polynucleotide encoding a signal
peptide (signal peptide element). The signal peptide may promote
surface trafficking of the introduced TCR or CAR. Exemplary signal
peptides include, but are not limited to CD8 signal peptide,
immunoglobulin signal peptides, where specific examples include
GM-CSF and IgG kappa. Such signal peptides are described in Trends
Biochem Sci. 2006 October; 31(10):563-71. Epub 2006 Aug. 21; and
An, et al. "Construction of a New Anti-CD19 Chimeric Antigen
Receptor and the Anti-Leukemia Function Study of the Transduced T
Cells." Oncotarget 7.9 (2016): 10638-10649. PMC. Web. 16 Aug. 2018;
which are hereby incorporated by reference.
[0558] In some cases, e.g., cases where the alpha and beta chains
are expressed from a single construct or open reading frame, or
cases wherein a marker gene is included in the construct, the
construct may comprise a ribosomal skip sequence. The ribosomal
skip sequence may be a 2A peptide, e.g., a P2A or T2A peptide.
Exemplary P2A and T2A peptides are described in Scientific Reports
volume 7, Article number: 2193 (2017), hereby incorporated by
reference in its entirety. In some cases, a FURIN/PACE cleavage
site is introduced upstream of the 2A element. FURIN/PACE cleavage
sites are described in, e.g.,
http://www.nuolan.net/substrates.html. The cleavage peptide may
also be a factor Xa cleavage site. In cases where the alpha and
beta chains are expressed from a single construct or open reading
frame, the construct may comprise an internal ribosome entry site
(IRES).
[0559] The construct may further comprise one or more marker genes.
Exemplary marker genes include but are not limited to GFP,
luciferase, HA, lacZ. The marker may be a selectable marker, such
as an antibiotic resistance marker, a heavy metal resistance
marker, or a biocide resistant marker, as is known to those of
skill in the art. The marker may be a complementation marker for
use in an auxotrophic host. Exemplary complementation markers and
auxotrophic hosts are described in Gene. 2001 Jan. 24;
263(1-2):159-69. Such markers may be expressed via an IRES, a
frameshift sequence, a 2A peptide linker, a fusion with the TCR or
CAR, or expressed separately from a separate promoter.
[0560] Exemplary vectors or systems for introducing TCRs or CARs
into recipient cells include, but are not limited to
Adeno-associated virus, Adenovirus, Adenovirus+Modified vaccinia,
Ankara virus (MVA), Adenovirus+Retrovirus, Adenovirus+Sendai virus,
Adenovirus+Vaccinia virus, Alphavirus (VEE) Replicon Vaccine,
Antisense oligonucleotide, Bifidobacterium longum, CRISPR-Cas9, E.
coli, Flavivirus, Gene gun, Herpesviruses, Herpes simplex virus,
Lactococcus lactis, Electroporation, Lentivirus, Lipofection,
Listeria monocytogenes, Measles virus, Modified Vaccinia Ankara
virus (MVA), mRNA Electroporation, Naked/Plasmid DNA, Naked/Plasmid
DNA+Adenovirus, Naked/Plasmid DNA+Modified Vaccinia Ankara virus
(MVA), Naked/Plasmid DNA+RNA transfer, Naked/Plasmid DNA+Vaccinia
virus, Naked/Plasmid DNA+Vesicular stomatitis virus, Newcastle
disease virus, Non-viral, PiggyBacm (PB) Transposon,
nanoparticle-based systems, Poliovirus, Poxvirus, Poxvirus+Vaccinia
virus, Retrovirus, RNA transfer, RNA transfer+Naked/Plasmid DNA,
RNA virus, Saccharomyces cerevisiae, Salmonella typhimurium,
Semliki forest virus, Sendai virus, Shigella dysenteriae, Simian
virus, siRNA, Sleeping Beauty transposon, Streptococcus mutans,
Vaccinia virus, Venezuelan equine encephalitis virus replicon,
Vesicular stomatitis virus, and Vibrio cholera.
[0561] In preferred embodiments, the TCR or CAR is introduced into
the recipient cell via adeno associated virus (AAV), adenovirus,
CRISPR-CAS9, herpesvirus, lentivirus, lipofection, mRNA
electroporation, PiggyBacm (PB) Transposon, retrovirus, RNA
transfer, or Sleeping Beauty transposon.
[0562] In some embodiments, a vector for introducing a TCR or CAR
into a recipient cell is a viral vector. Exemplary viral vectors
include adenoviral vectors, adeno-associated viral (AAV) vectors,
lentiviral vectors, herpes viral vectors, retroviral vectors, and
the like. Such vectors are described herein.
[0563] Exemplary embodiments of TCR constructs for introducing a
TCR or CAR into recipient cells is shown in FIG. 2. In some
embodiments, a TCR construct includes, from the 5'-3' direction,
the following polynucleotide sequences: a promoter sequence, a
signal peptide sequence, a TCR .beta. variable (TCR.beta.v)
sequence, a TCR .beta. constant ((TCR.beta.c) sequence, a cleavage
peptide (e.g., P2A), a signal peptide sequence, a TCR .alpha.
variable (TCR.alpha.v) sequence, and a TCR .alpha. constant
(TCR.alpha.c) sequence. In some embodiments, the TCR.beta.c and
TCR.alpha.c sequences of the construct include one or more murine
regions, e.g., full murine constant sequences or
human.fwdarw.murine amino acid exchanges as described herein. In
some embodiments, the construct further includes, 3' of the
TCR.alpha.c sequence, a cleavage peptide sequence (e.g., T2A)
followed by a reporter gene. In an embodiment, the construct
includes, from the 5'-3' direction, the following polynucleotide
sequences: a promoter sequence, a signal peptide sequence, a TCR
.beta. variable (TCR.beta.v) sequence, a TCR .beta. constant
((TCR.beta.c) sequence containing one or more murine regions, a
cleavage peptide (e.g., P2A), a signal peptide sequence, a TCR
.alpha. variable (TCR.alpha.v) sequence, and a TCR .alpha. constant
(TCR.alpha.c) sequence containing one or more murine regions, a
cleavage peptide (e.g., T2A), and a reporter gene.
[0564] FIG. 3 depicts an exemplary construct backbone sequence for
cloning TCRs into expression systems for therapy development.
[0565] FIG. 4 depicts an exemplary construct sequence for cloning
an identified A*0201_LLASSILCA-specific TCR into expression systems
for therapy development.
[0566] FIG. 5 depicts an exemplary construct sequence for cloning
an identified A*0101_EVDPIGHLY-specific TCR into expression systems
for therapy development.
[0567] Nucleotides, Vectors, Host Cells, and Related Methods
[0568] Also provided are isolated nucleic acids encoding
HLA-PEPTIDE ABPs, vectors comprising the nucleic acids, and host
cells comprising the vectors and nucleic acids, as well as
recombinant techniques for the production of the ABPs.
[0569] The nucleic acids may be recombinant. The recombinant
nucleic acids may be constructed outside living cells by joining
natural or synthetic nucleic acid segments to nucleic acid
molecules that can replicate in a living cell, or replication
products thereof. For purposes herein, the replication can be in
vitro replication or in vivo replication.
[0570] For recombinant production of an ABP, the nucleic acid(s)
encoding it may be isolated and inserted into a replicable vector
for further cloning (i.e., amplification of the DNA) or expression.
In some aspects, the nucleic acid may be produced by homologous
recombination, for example as described in U.S. Pat. No. 5,204,244,
incorporated by reference in its entirety.
[0571] Many different vectors are known in the art. The vector
components generally include one or more of the following: a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence, for example as described in U.S. Pat. No. 5,534,615,
incorporated by reference in its entirety.
[0572] Exemplary vectors or constructs suitable for expressing an
ABP, e.g., a TCR, CAR, antibody, or antigen binding fragment
thereof, include, e.g., the pUC series (Fermentas Life Sciences),
the pBluescript series (Stratagene, LaJolla, Calif.), the pET
series (Novagen, Madison, Wis.), the pGEX series (Pharmacia
Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto,
Calif.). Bacteriophage vectors, such as AGTIO, AGT1 1, AZapII
(Stratagene), AEMBL4, and ANM1 149, are also suitable for
expressing an ABP disclosed herein.
[0573] Illustrative examples of suitable host cells are provided
below. These host cells are not meant to be limiting, and any
suitable host cell may be used to produce the ABPs provided
herein.
[0574] Suitable host cells include any prokaryotic (e.g.,
bacterial), lower eukaryotic (e.g., yeast), or higher eukaryotic
(e.g., mammalian) cells. Suitable prokaryotes include eubacteria,
such as Gram-negative or Gram-positive organisms, for example,
Enterobacteriaceae such as Escherichia (E. coli), Enterobacter,
Erwinia, Klebsiella, Proteus, Salmonella (S. typhimurium), Serratia
(S. marcescans), Shigella, Bacilli (B. subtilis and B.
lichenformis), Pseudomonas (P. aeruginosa), and Streptomyces. One
useful E. coli cloning host is E. coli 294, although other strains
such as E. coli B, E. coli X1776, and E. coli W3110 are also
suitable.
[0575] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are also suitable cloning or expression
hosts for HLA-PEPTIDE ABP-encoding vectors. Saccharomyces
cerevisiae, or common baker's yeast, is a commonly used lower
eukaryotic host microorganism. However, a number of other genera,
species, and strains are available and useful, such as
Schizosaccharomyces pombe, Kluyveromyces (K. lactis, K. fragilis,
K. bulgaricus K. wickeramii, K. waltii, K. drosophilarum, K.
thermotolerans, and K. marxianus), Yarrowia, Pichia pastoris,
Candida (C. albicans), Trichoderma reesia, Neurospora crassa,
Schwanniomyces (S. occidentalis), and filamentous fungi such as,
for example Penicillium, Tolypocladium, and Aspergillus (A.
nidulans and A. niger).
[0576] Useful mammalian host cells include COS-7 cells, HEK293
cells; baby hamster kidney (BHK) cells; Chinese hamster ovary
(CHO); mouse sertoli cells; African green monkey kidney cells
(VERO-76), and the like.
[0577] The host cells used to produce the HLA-PEPTIDE ABP may be
cultured in a variety of media. Commercially available media such
as, for example, Ham's F10, Minimal Essential Medium (MEM),
RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM) are
suitable for culturing the host cells. In addition, any of the
media described in Ham et al., Meth. Enz., 1979, 58:44; Barnes et
al., Anal. Biochem., 1980, 102:255; and U.S. Pat. Nos. 4,767,704,
4,657,866, 4,927,762, 4,560,655, and 5,122,469; or WO 90/03430 and
WO 87/00195 may be used. Each of the foregoing references is
incorporated by reference in its entirety.
[0578] Any of these media may be supplemented as necessary with
hormones and/or other growth factors (such as insulin, transferrin,
or epidermal growth factor), salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleotides (such as adenosine and thymidine), antibiotics, trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art.
[0579] The culture conditions, such as temperature, pH, and the
like, are those previously used with the host cell selected for
expression, and will be apparent to the ordinarily skilled
artisan.
[0580] When using recombinant techniques, the ABP can be produced
intracellularly, in the periplasmic space, or directly secreted
into the medium. If the ABP is produced intracellularly, as a first
step, the particulate debris, either host cells or lysed fragments,
is removed, for example, by centrifugation or ultrafiltration. For
example, Carter et al. (Bio/Technology, 1992, 10:163-167,
incorporated by reference in its entirety) describes a procedure
for isolating ABPs which are secreted to the periplasmic space of
E. coli. Briefly, cell paste is thawed in the presence of sodium
acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF)
over about 30 min. Cell debris can be removed by
centrifugation.
[0581] In some embodiments, the ABP is produced in a cell-free
system. In some aspects, the cell-free system is an in vitro
transcription and translation system as described in Yin et al.,
mAbs, 2012, 4:217-225, incorporated by reference in its entirety.
In some aspects, the cell-free system utilizes a cell-free extract
from a eukaryotic cell or from a prokaryotic cell. In some aspects,
the prokaryotic cell is E. coli. Cell-free expression of the ABP
may be useful, for example, where the ABP accumulates in a cell as
an insoluble aggregate, or where yields from periplasmic expression
are low.
[0582] Where the ABP is secreted into the medium, supernatants from
such expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon.RTM. or Millipore.RTM. Pellcon.RTM. ultrafiltration unit.
A protease inhibitor such as PMSF may be included in any of the
foregoing steps to inhibit proteolysis and antibiotics may be
included to prevent the growth of adventitious contaminants.
[0583] The ABP composition prepared from the cells can be purified
using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being a particularly useful purification
technique. The suitability of protein A as an affinity ligand
depends on the species and isotype of any immunoglobulin Fc domain
that is present in the ABP. Protein A can be used to purify ABPs
that comprise human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth., 1983, 62:1-13, incorporated by
reference in its entirety). Protein G is useful for all mouse
isotypes and for human 73 (Guss et al., EMBO J., 1986, 5:1567-1575,
incorporated by reference in its entirety).
[0584] The matrix to which the affinity ligand is attached is most
often agarose, but other matrices are available. Mechanically
stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the ABP
comprises a CH3 domain, the BakerBond ABX.RTM. resin is useful for
purification.
[0585] Other techniques for protein purification, such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin Sepharose.RTM., chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available, and can be applied by one
of skill in the art.
[0586] Following any preliminary purification step(s), the mixture
comprising the ABP of interest and contaminants may be subjected to
low pH hydrophobic interaction chromatography using an elution
buffer at a pH between about 2.5 to about 4.5, generally performed
at low salt concentrations (e.g., from about 0 to about 0.25 M
salt).
[0587] Methods of Making HLA-PEPTIDE ABPs
HLA-PEPTIDE Antigen Preparation
[0588] The HLA-PEPTIDE antigen used for isolation or creation of
the ABPs provided herein may be intact HLA-PEPTIDE or a fragment of
HLA-PEPTIDE. The HLA-PEPTIDE antigen may be, for example, in the
form of isolated protein or a protein expressed on the surface of a
cell.
[0589] In some embodiments, the HLA-PEPTIDE antigen is a
non-naturally occurring variant of HLA-PEPTIDE, such as a
HLA-PEPTIDE protein having an amino acid sequence or
post-translational modification that does not occur in nature.
[0590] In some embodiments, the HLA-PEPTIDE antigen is truncated by
removal of, for example, intracellular or membrane-spanning
sequences, or signal sequences. In some embodiments, the
HLA-PEPTIDE antigen is fused at its C-terminus to a human IgG1 Fc
domain or a polyhistidine tag.
Methods of Identifying ABPs
[0591] ABPs that bind HLA-PEPTIDE can be identified using any
method known in the art, e.g., phage display or immunization of a
subject.
[0592] One method of identifying an antigen binding protein
includes providing at least one HLA-PEPTIDE target; and binding the
at least one target with an antigen binding protein, thereby
identifying the antigen binding protein. The antigen binding
protein can be present in a library comprising a plurality of
distinct antigen binding proteins.
[0593] In some embodiments, the library is a phage display library.
The phage display library can be developed so that it is
substantially free of antigen binding proteins that
non-specifically bind the HLA of the HLA-PEPTIDE target. The
antigen binding protein can be present in a yeast display library
comprising a plurality of distinct antigen binding proteins. The
yeast display library can be developed so that it is substantially
free of antigen binding proteins that non-specifically bind the HLA
of the HLA-PEPTIDE target.
[0594] In some embodiments, the library is a yeast display
library.
[0595] In some embodiments, the library is a TCR display library.
Exemplary TCR display libraries and methods of using such TCR
display libraries are described in WO 98/39482; WO 01/62908; WO
2004/044004; WO2005116646, WO2014018863, WO2015136072,
WO2017046198; and Helmut et al, (2000) PNAS 97 (26) 14578-14583,
which are hereby incorporated by reference in their entirety.
[0596] In some aspects, the binding step is performed more than
once, optionally at least three times, e.g., at least 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10.times..
[0597] In addition, the method can also include contacting the
antigen binding protein with one or more peptide-HLA complexes that
are distinct from the HLA-PEPTIDE target to determine if the
antigen binding protein selectively binds the HLA-PEPTIDE
target.
[0598] Another method of identifying an antigen binding protein can
include obtaining at least one HLA-PEPTIDE target; administering
the HLA-PEPTIDE target to a subject (e.g., a mouse, rabbit or a
llama), optionally in combination with an adjuvant; and isolating
the antigen binding protein from the subject. Isolating the antigen
binding protein can include screening the serum of the subject to
identify the antigen binding protein. The method can also include
contacting the antigen binding protein with one or more peptide-HLA
complexes that are distinct from the HLA-PEPTIDE target, e.g., to
determine if the antigen binding protein selectively binds to the
HLA-PEPTIDE target. An antigen binding protein that is identified
can be humanized.
[0599] In some aspects, isolating the antigen binding protein
comprises isolating a B cell from the subject that expresses the
antigen binding protein. The B cell can be used to create a
hybridoma. The B cell can also be used for cloning one or more of
its CDRs. The B cell can also be immortalized, for example, by
using EBV transformation. Sequences encoding an antigen binding
protein can be cloned from immortalized B cells or can be cloned
directly from B cells isolated from an immunized subject. A library
that comprises the antigen binding protein of the B cell can also
be created, optionally wherein the library is phage display or
yeast display.
[0600] Another method of identifying an antigen binding protein can
include obtaining a cell comprising the antigen binding protein;
contacting the cell with an HLA-multimer (e.g., a tetramer)
comprising at least one HLA-PEPTIDE target; and identifying the
antigen binding protein via binding between the HLA-multimer and
the antigen binding protein.
[0601] The cell can be, e.g., a T cell, optionally a cytotoxic T
lymphocyte (CTL), or a natural killer (NK) cell, for example. The
method can further include isolating the cell, optionally using
flow cytometry, magnetic separation, or single cell separation. The
method can further include sequencing the antigen binding
protein.
[0602] Another method of identifying an antigen binding protein can
include obtaining one or more cells comprising the antigen binding
protein; activating the one or more cells with at least one
HLA-PEPTIDE target presented on at least one antigen presenting
cell (APC); and identifying the antigen binding protein via
selection of one or more cells activated by interaction with at
least one HLA-PEPTIDE target.
[0603] The cell can be, e.g., a T cell, optionally a CTL, or an NK
cell, for example. The method can further include isolating the
cell, optionally using flow cytometry, magnetic separation, or
single cell separation. The method can further include sequencing
the antigen binding protein.
Methods of Making Monoclonal ABPs
[0604] Monoclonal ABPs may be obtained, for example, using the
hybridoma method first described by Kohler et al., Nature, 1975,
256:495-497 (incorporated by reference in its entirety), and/or by
recombinant DNA methods (see e.g., U.S. Pat. No. 4,816,567,
incorporated by reference in its entirety). Monoclonal ABPs may
also be obtained, for example, using phage or yeast-based
libraries. See e.g., U.S. Pat. Nos. 8,258,082 and 8,691,730, each
of which is incorporated by reference in its entirety.
[0605] In the hybridoma method, a mouse or other appropriate host
animal is immunized to elicit lymphocytes that produce or are
capable of producing ABPs that will specifically bind to the
protein used for immunization. Alternatively, lymphocytes may be
immunized in vitro. Lymphocytes are then fused with myeloma cells
using a suitable fusing agent, such as polyethylene glycol, to form
a hybridoma cell. See Goding J. W., Monoclonal ABPs: Principles and
Practice 3.sup.rd ed. (1986) Academic Press, San Diego, Calif.,
incorporated by reference in its entirety.
[0606] The hybridoma cells are seeded and grown in a suitable
culture medium that contains one or more substances that inhibit
the growth or survival of the unfused, parental myeloma cells. For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine (HAT medium), which substances prevent
the growth of HGPRT-deficient cells.
[0607] Useful myeloma cells are those that fuse efficiently,
support stable high-level production of ABP by the selected
ABP-producing cells, and are sensitive media conditions, such as
the presence or absence of HAT medium. Among these, preferred
myeloma cell lines are murine myeloma lines, such as those derived
from MOPC-21 and MC-11 mouse tumors (available from the Salk
Institute Cell Distribution Center, San Diego, Calif.), and SP-2 or
X63-Ag8-653 cells (available from the American Type Culture
Collection, Rockville, Md.). Human myeloma and mouse-human
heteromyeloma cell lines also have been described for the
production of human monoclonal ABPs. See e.g., Kozbor, J. Immunol.,
1984, 133:3001, incorporated by reference in its entirety.
[0608] After the identification of hybridoma cells that produce
ABPs of the desired specificity, affinity, and/or biological
activity, selected clones may be subcloned by limiting dilution
procedures and grown by standard methods. See Goding, supra.
Suitable culture media for this purpose include, for example, D-MEM
or RPMI-1640 medium. In addition, the hybridoma cells may be grown
in vivo as ascites tumors in an animal.
[0609] DNA encoding the monoclonal ABPs may be readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal ABPs).
Thus, the hybridoma cells can serve as a useful source of DNA
encoding ABPs with the desired properties. Once isolated, the DNA
may be placed into expression vectors, which are then transfected
into host cells such as bacteria (e.g., E. coli), yeast (e.g.,
Saccharomyces or Pichia sp.), COS cells, Chinese hamster ovary
(CHO) cells, or myeloma cells that do not otherwise produce ABP, to
produce the monoclonal ABPs.
Methods of Making Chimeric ABPs
[0610] Illustrative methods of making chimeric ABPs are described,
for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc.
Natl. Acad. Sci. USA, 1984, 81:6851-6855; each of which is
incorporated by reference in its entirety. In some embodiments, a
chimeric ABP is made by using recombinant techniques to combine a
non-human variable region (e.g., a variable region derived from a
mouse, rat, hamster, rabbit, or non-human primate, such as a
monkey) with a human constant region.
Methods of Making Humanized ABPs
[0611] Humanized ABPs may be generated by replacing most, or all,
of the structural portions of a non-human monoclonal ABP with
corresponding human ABP sequences. Consequently, a hybrid molecule
is generated in which only the antigen-specific variable, or CDR,
is composed of non-human sequence. Methods to obtain humanized ABPs
include those described in, for example, Winter and Milstein,
Nature, 1991, 349:293-299; Rader et al., Proc. Nat. Acad. Sci.
U.S.A., 1998, 95:8910-8915; Steinberger et al., J. Biol. Chem.,
2000, 275:36073-36078; Queen et al., Proc. Natl. Acad. Sci. U.S.A.,
1989, 86:10029-10033; and U.S. Pat. Nos. 5,585,089, 5,693,761,
5,693,762, and 6,180,370; each of which is incorporated by
reference in its entirety.
Methods of Making Human ABPs
[0612] Human ABPs can be generated by a variety of techniques known
in the art, for example by using transgenic animals (e.g.,
humanized mice). See, e.g., Jakobovits et al., Proc. Natl. Acad.
Sci. U.S.A., 1993, 90:2551; Jakobovits et al., Nature, 1993,
362:255-258; Bruggermann et al., Year in Immuno., 1993, 7:33; and
U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807; each of which is
incorporated by reference in its entirety. Human ABPs can also be
derived from phage-display libraries (see e.g., Hoogenboom et al.,
J. Mol. Biol., 1991, 227:381-388; Marks et al., J. Mol. Biol.,
1991, 222:581-597; and U.S. Pat. Nos. 5,565,332 and 5,573,905; each
of which is incorporated by reference in its entirety). Human ABPs
may also be generated by in vitro activated B cells (see e.g., U.S.
Pat. Nos. 5,567,610 and 5,229,275, each of which is incorporated by
reference in its entirety). Human ABPs may also be derived from
yeast-based libraries (see e.g., U.S. Pat. No. 8,691,730,
incorporated by reference in its entirety).
Methods of Making ABP Fragments
[0613] The ABP fragments provided herein may be made by any
suitable method, including the illustrative methods described
herein or those known in the art. Suitable methods include
recombinant techniques and proteolytic digestion of whole ABPs.
Illustrative methods of making ABP fragments are described, for
example, in Hudson et al., Nat. Med., 2003, 9:129-134, incorporated
by reference in its entirety. Methods of making scFv ABPs are
described, for example, in Pluckthun, in The Pharmacology of
Monoclonal ABPs, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994); WO 93/16185; and
U.S. Pat. Nos. 5,571,894 and 5,587,458; each of which is
incorporated by reference in its entirety.
Methods of Making Alternative Scaffolds
[0614] The alternative scaffolds provided herein may be made by any
suitable method, including the illustrative methods described
herein or those known in the art. For example, methods of preparing
Adnectins.TM. are described in Emanuel et al., mAbs, 2011, 3:38-48,
incorporated by reference in its entirety. Methods of preparing
iMabs are described in U.S. Pat. Pub. No. 2003/0215914,
incorporated by reference in its entirety. Methods of preparing
Anticalins.RTM. are described in Vogt and Skerra, Chem. Biochem.,
2004, 5:191-199, incorporated by reference in its entirety. Methods
of preparing Kunitz domains are described in Wagner et al.,
Biochem. & Biophys. Res. Comm., 1992, 186:118-1145,
incorporated by reference in its entirety. Methods of preparing
thioredoxin peptide aptamers are provided in Geyer and Brent, Meth.
Enzymol., 2000, 328:171-208, incorporated by reference in its
entirety. Methods of preparing Affibodies are provided in
Fernandez, Curr. Opinion in Biotech., 2004, 15:364-373,
incorporated by reference in its entirety. Methods of preparing
DARPins are provided in Zahnd et al., J. Mol. Biol., 2007,
369:1015-1028, incorporated by reference in its entirety. Methods
of preparing Affilins are provided in Ebersbach et al., J. Mol.
Biol., 2007, 372:172-185, incorporated by reference in its
entirety. Methods of preparing Tetranectins are provided in
Graversen et al., J. Biol. Chem., 2000, 275:37390-37396,
incorporated by reference in its entirety. Methods of preparing
Avimers are provided in Silverman et al., Nature Biotech., 2005,
23:1556-1561, incorporated by reference in its entirety. Methods of
preparing Fynomers are provided in Silacci et al., J Biol. Chem.,
2014, 289:14392-14398, incorporated by reference in its entirety.
Further information on alternative scaffolds is provided in Binz et
al., Nat. Biotechnol., 2005 23:1257-1268; and Skerra, Current Opin.
in Biotech., 2007 18:295-304, each of which is incorporated by
reference in its entirety.
Methods of Making Multispecific ABPs
[0615] The multispecific ABPs provided herein may be made by any
suitable method, including the illustrative methods described
herein or those known in the art. Methods of making common light
chain ABPs are described in Merchant et al., Nature Biotechnol.,
1998, 16:677-681, incorporated by reference in its entirety.
Methods of making tetravalent bispecific ABPs are described in
Coloma and Morrison, Nature Biotechnol., 1997, 15:159-163,
incorporated by reference in its entirety. Methods of making hybrid
immunoglobulins are described in Milstein and Cuello, Nature, 1983,
305:537-540; and Staerz and Bevan, Proc. Natl. Acad. Sci. USA,
1986, 83:1453-1457; each of which is incorporated by reference in
its entirety. Methods of making immunoglobulins with
knobs-into-holes modification are described in U.S. Pat. No.
5,731,168, incorporated by reference in its entirety. Methods of
making immunoglobulins with electrostatic modifications are
provided in WO 2009/089004, incorporated by reference in its
entirety. Methods of making bispecific single chain ABPs are
described in Traunecker et al., EMBO J., 1991, 10:3655-3659; and
Gruber et al., J. Immunol., 1994, 152:5368-5374; each of which is
incorporated by reference in its entirety. Methods of making
single-chain ABPs, whose linker length may be varied, are described
in U.S. Pat. Nos. 4,946,778 and 5,132,405, each of which is
incorporated by reference in its entirety. Methods of making
diabodies are described in Hollinger et al., Proc. Natl. Acad. Sci.
USA, 1993, 90:6444-6448, incorporated by reference in its entirety.
Methods of making triabodies and tetrabodies are described in
Todorovska et al., J. Immunol. Methods, 2001, 248:47-66,
incorporated by reference in its entirety. Methods of making
trispecific F(ab')3 derivatives are described in Tutt et al. J.
Immunol., 1991, 147:60-69, incorporated by reference in its
entirety. Methods of making cross-linked ABPs are described in U.S.
Pat. No. 4,676,980; Brennan et al., Science, 1985, 229:81-83;
Staerz, et al. Nature, 1985, 314:628-631; and EP 0453082; each of
which is incorporated by reference in its entirety. Methods of
making antigen-binding domains assembled by leucine zippers are
described in Kostelny et al., J. Immunol., 1992, 148:1547-1553,
incorporated by reference in its entirety. Methods of making ABPs
via the DNL approach are described in U.S. Pat. Nos. 7,521,056;
7,550,143; 7,534,866; and 7,527,787; each of which is incorporated
by reference in its entirety. Methods of making hybrids of ABP and
non-ABP molecules are described in WO 93/08829, incorporated by
reference in its entirety, for examples of such ABPs. Methods of
making DAF ABPs are described in U.S. Pat. Pub. No. 2008/0069820,
incorporated by reference in its entirety. Methods of making ABPs
via reduction and oxidation are described in Carlring et al., PLoS
One, 2011, 6:e22533, incorporated by reference in its entirety.
Methods of making DVD-Igs' are described in U.S. Pat. No.
7,612,181, incorporated by reference in its entirety. Methods of
making DARTs.TM. are described in Moore et al., Blood, 2011,
117:454-451, incorporated by reference in its entirety. Methods of
making DuoBodies.RTM. are described in Labrijn et al., Proc. Natd.
Acad. Sci. USA, 2013, 110:5145-5150; Gramer et al., mAbs, 2013,
5:962-972; and Labrijn et al., Nature Protocols, 2014, 9:2450-2463;
each of which is incorporated by reference in its entirety. Methods
of making ABPs comprising scFvs fused to the C-terminus of the CH3
from an IgG are described in Coloma and Morrison, Nature
Biotechnol., 1997, 15:159-163, incorporated by reference in its
entirety. Methods of making ABPs in which a Fab molecule is
attached to the constant region of an immunoglobulin are described
in Miler et al., J. Immunol., 2003, 170:4854-4861, incorporated by
reference in its entirety. Methods of making CovX-Bodies are
described in Doppalapudi et al., Proc. Natl. Acad. Sci. USA, 2010,
107:22611-22616, incorporated by reference in its entirety. Methods
of making Fcab ABPs are described in Wozniak-Knopp et al., Protein
Eng. Des. Sel., 2010, 23:289-297, incorporated by reference in its
entirety. Methods of making TandAb.RTM. ABPs are described in
Kipriyanov et al., J. Mol. Biol., 1999, 293:41-56 and Zhukovsky et
al., Blood, 2013, 122:5116, each of which is incorporated by
reference in its entirety. Methods of making tandem Fabs are
described in WO 2015/103072, incorporated by reference in its
entirety. Methods of making Zybodies' are described in LaFleur et
al., mAbs, 2013, 5:208-218, incorporated by reference in its
entirety.
Methods of Making Variants
[0616] Any suitable method can be used to introduce variability
into a polynucleotide sequence(s) encoding an ABP, including
error-prone PCR, chain shuffling, and oligonucleotide-directed
mutagenesis such as trinucleotide-directed mutagenesis (TRIM). In
some aspects, several CDR residues (e.g., 4-6 residues at a time)
are randomized. CDR residues involved in antigen binding may be
specifically identified, for example, using alanine scanning
mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often
targeted for mutation.
[0617] The introduction of diversity into the variable regions
and/or CDRs can be used to produce a secondary library. The
secondary library is then screened to identify ABP variants with
improved affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, for
example, in Hoogenboom et al., Methods in Molecular Biology, 2001,
178:1-37, incorporated by reference in its entirety.
[0618] Methods for Engineering Cells with ABPs
[0619] Also provided are methods, nucleic acids, compositions, and
kits, for expressing the ABPs, including receptors comprising
antibodies, CARs, and TCRs, and for producing genetically
engineered cells expressing such ABPs. The genetic engineering
generally involves introduction of a nucleic acid encoding the
recombinant or engineered component into the cell, such as by
retroviral transduction, transfection, or transformation.
[0620] In some embodiments, gene transfer is accomplished by first
stimulating the cell, such as by combining it with a stimulus that
induces a response such as proliferation, survival, and/or
activation, e.g., as measured by expression of a cytokine or
activation marker, followed by transduction of the activated cells,
and expansion in culture to numbers sufficient for clinical
applications.
[0621] In some contexts, overexpression of a stimulatory factor
(for example, a lymphokine or a cytokine) may be toxic to a
subject. Thus, in some contexts, the engineered cells include gene
segments that cause the cells to be susceptible to negative
selection in vivo, such as upon administration in adoptive
immunotherapy. For example in some aspects, the cells are
engineered so that they can be eliminated as a result of a change
in the in vivo condition of the patient to which they are
administered. The negative selectable phenotype may result from the
insertion of a gene that confers sensitivity to an administered
agent, for example, a compound. Negative selectable genes include
the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene
(Wigler et al., Cell II: 223, 1977) which confers ganciclovir
sensitivity; the cellular hypoxanthine phosphribosyltransferase
(HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT)
gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl.
Acad. Sci. USA. 89:33 (1992)).
[0622] In some aspects, the cells further are engineered to promote
expression of cytokines or other factors. Various methods for the
introduction of genetically engineered components, e.g., antigen
receptors, e.g., CARs, are well known and may be used with the
provided methods and compositions. Exemplary methods include those
for transfer of nucleic acids encoding the receptors, including via
viral, e.g., retroviral or lentiviral, transduction, transposons,
and electroporation.
[0623] In some embodiments, recombinant nucleic acids are
transferred into cells using recombinant infectious virus
particles, such as, e.g., vectors derived from simian virus 40
(SV40), adenoviruses, adeno-associated virus (AAV). In some
embodiments, recombinant nucleic acids are transferred into T cells
using recombinant lentiviral vectors or retroviral vectors, such as
gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene
Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000)
Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther
Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov.
29(11): 550-557.
[0624] In some embodiments, the retroviral vector has a long
terminal repeat sequence (LTR), e.g., a retroviral vector derived
from the Moloney murine leukemia virus (MoMLV), myeloproliferative
sarcoma virus (MPSV), murine embryonic stem cell virus (MESV),
murine stem cell virus (MSCV), spleen focus forming virus (SFFV),
or adeno-associated virus (AAV). Most retroviral vectors are
derived from murine retroviruses. In some embodiments, the
retroviruses include those derived from any avian or mammalian cell
source. The retroviruses typically are amphotropic, meaning that
they are capable of infecting host cells of several species,
including humans. In one embodiment, the gene to be expressed
replaces the retroviral gag, pol and/or env sequences. A number of
illustrative retroviral systems have been described (e.g., U.S.
Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)
BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy
1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al.
(1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie
and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
[0625] Methods of lentiviral transduction are known. Exemplary
methods are described in, e.g., Wang et al. (2012) J. Immunother.
35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644;
Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and
Cavalieri et al. (2003) Blood. 102(2): 497-505.
[0626] In some embodiments, recombinant nucleic acids are
transferred into T cells via electroporation (see, e.g., Chicaybam
et al, (2013) PLoS ONE 8(3): e60298; Van Tedeloo et al. (2000) Gene
Therapy 7(16): 1431-1437; and Roth et al. (2018) Nature
559:405-409). In some embodiments, recombinant nucleic acids are
transferred into T cells via transposition (see, e.g., Manuri et
al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec
Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol
506: 115-126). Other methods of introducing and expressing genetic
material in immune cells include calcium phosphate transfection
(e.g., as described in Current Protocols in Molecular Biology, John
Wiley & Sons, New York. N.Y.), protoplast fusion, cationic
liposome-mediated transfection; tungsten particle-facilitated
microparticle bombardment (Johnston, Nature, 346: 776-777 (1990));
and strontium phosphate DNA co-precipitation (Brash et al., Mol.
Cell Biol., 7: 2031-2034 (1987)).
[0627] Other approaches and vectors for transfer of the nucleic
acids encoding the recombinant products are those described, e.g.,
in international patent application, Publication No.: WO2014055668,
and U.S. Pat. No. 7,446,190.
[0628] Among additional nucleic acids, e.g., genes for introduction
are those to improve the efficacy of therapy, such as by promoting
viability and/or function of transferred cells; genes to provide a
genetic marker for selection and/or evaluation of the cells, such
as to assess in vivo survival or localization; genes to improve
safety, for example, by making the cell susceptible to negative
selection in vivo as described by Lupton S. D. et al., Mol. and
Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy
3:319-338 (1992); see also the publications of PCT/US91/08442 and
PCT/US94/05601 by Lupton et al. describing the use of bifunctional
selectable fusion genes derived from fusing a dominant positive
selectable marker with a negative selectable marker. See, e.g.,
Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
[0629] Preparation of Engineered Cells
[0630] In some embodiments, preparation of the engineered cells
includes one or more culture and/or preparation steps. The cells
for introduction of the HLA-PEPTIDE-ABP, e.g., TCR or CAR, can be
isolated from a sample, such as a biological sample, e.g., one
obtained from or derived from a subject. In some embodiments, the
subject from which the cell is isolated is one having the disease
or condition or in need of a cell therapy or to which cell therapy
will be administered. The subject in some embodiments is a human in
need of a particular therapeutic intervention, such as the adoptive
cell therapy for which cells are being isolated, processed, and/or
engineered.
[0631] Accordingly, the cells in some embodiments are primary
cells, e.g., primary human cells. The samples include tissue,
fluid, and other samples taken directly from the subject, as well
as samples resulting from one or more processing steps, such as
separation, centrifugation, genetic engineering (e.g. transduction
with viral vector), washing, and/or incubation. The biological
sample can be a sample obtained directly from a biological source
or a sample that is processed. Biological samples include, but are
not limited to, body fluids, such as blood, plasma, serum,
cerebrospinal fluid, synovial fluid, urine and sweat, tissue and
organ samples, including processed samples derived therefrom.
[0632] In some aspects, the sample from which the cells are derived
or isolated is blood or a blood-derived sample, or is or is derived
from an apheresis or leukapheresis product. Exemplary samples
include whole blood, peripheral blood mononuclear cells (PBMCs),
leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia,
lymphoma, lymph node, gut associated lymphoid tissue, mucosa
associated lymphoid tissue, spleen, other lymphoid tissues, liver,
lung, stomach, intestine, colon, kidney, pancreas, breast, bone,
prostate, cervix, testes, ovaries, tonsil, or other organ, and/or
cells derived therefrom. Samples include, in the context of cell
therapy, e.g., adoptive cell therapy, samples from autologous and
allogeneic sources.
[0633] In some embodiments, the cells are derived from cell lines,
e.g., T cell lines. The cells in some embodiments are obtained from
a xenogeneic source, for example, from mouse, rat, non-human
primate, or pig.
[0634] In some embodiments, isolation of the cells includes one or
more preparation and/or non-affinity based cell separation steps.
In some examples, cells are washed, centrifuged, and/or incubated
in the presence of one or more reagents, for example, to remove
unwanted components, enrich for desired components, lyse or remove
cells sensitive to particular reagents. In some examples, cells are
separated based on one or more property, such as density, adherent
properties, size, sensitivity and/or resistance to particular
components.
[0635] In some examples, cells from the circulating blood of a
subject are obtained, e.g., by apheresis or leukapheresis. The
samples, in some aspects, contain lymphocytes, including T cells,
monocytes, granulocytes, B cells, other nucleated white blood
cells, red blood cells, and/or platelets, and in some aspects
contains cells other than red blood cells and platelets.
[0636] In some embodiments, the blood cells collected from the
subject are washed, e.g., to remove the plasma fraction and to
place the cells in an appropriate buffer or media for subsequent
processing steps. In some embodiments, the cells are washed with
phosphate buffered saline (PBS). In some embodiments, the wash
solution lacks calcium and/or magnesium and/or many or all divalent
cations. In some aspects, a washing step is accomplished a
semi-automated "flow-through" centrifuge (for example, the Cobe
2991 cell processor, Baxter) according to the manufacturer's
instructions. In some aspects, a washing step is accomplished by
tangential flow filtration (TFF) according to the manufacturer's
instructions. In some embodiments, the cells are resuspended in a
variety of biocompatible buffers after washing, such as, for
example, Ca++/Mg++ free PBS. In certain embodiments, components of
a blood cell sample are removed and the cells directly resuspended
in culture media.
[0637] In some embodiments, the methods include density-based cell
separation methods, such as the preparation of white blood cells
from peripheral blood by lysing the red blood cells and
centrifugation through a Percoll or Ficoll gradient.
[0638] In some embodiments, the isolation methods include the
separation of different cell types based on the expression or
presence in the cell of one or more specific molecules, such as
surface markers, e.g., surface proteins, intracellular markers, or
nucleic acid. In some embodiments, any known method for separation
based on such markers may be used. In some embodiments, the
separation is affinity- or immunoaffinity-based separation. For
example, the isolation in some aspects includes separation of cells
and cell populations based on the cells' expression or expression
level of one or more markers, typically cell surface markers, for
example, by incubation with an antibody or binding partner that
specifically binds to such markers, followed generally by washing
steps and separation of cells having bound the antibody or binding
partner, from those cells having not bound to the antibody or
binding partner.
[0639] Such separation steps can be based on positive selection, in
which the cells having bound the reagents are retained for further
use, and/or negative selection, in which the cells having not bound
to the antibody or binding partner are retained. In some examples,
both fractions are retained for further use. In some aspects,
negative selection can be particularly useful where no antibody is
available that specifically identifies a cell type in a
heterogeneous population, such that separation is best carried out
based on markers expressed by cells other than the desired
population.
[0640] The separation need not result in 100% enrichment or removal
of a particular cell population or cells expressing a particular
marker. For example, positive selection of or enrichment for cells
of a particular type, such as those expressing a marker, refers to
increasing the number or percentage of such cells, but need not
result in a complete absence of cells not expressing the marker.
Likewise, negative selection, removal, or depletion of cells of a
particular type, such as those expressing a marker, refers to
decreasing the number or percentage of such cells, but need not
result in a complete removal of all such cells.
[0641] In some examples, multiple rounds of separation steps are
carried out, where the positively or negatively selected fraction
from one step is subjected to another separation step, such as a
subsequent positive or negative selection. In some examples, a
single separation step can deplete cells expressing multiple
markers simultaneously, such as by incubating cells with a
plurality of antibodies or binding partners, each specific for a
marker targeted for negative selection. Likewise, multiple cell
types can simultaneously be positively selected by incubating cells
with a plurality of antibodies or binding partners expressed on the
various cell types.
[0642] For example, in some aspects, specific subpopulations of T
cells, such as cells positive or expressing high levels of one or
more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+,
CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by
positive or negative selection techniques.
[0643] For example, CD3+, CD28+ T cells can be positively selected
using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS.RTM.
M-450 CD3/CD28 T Cell Expander).
[0644] In some embodiments, isolation is carried out by enrichment
for a particular cell population by positive selection, or
depletion of a particular cell population, by negative selection.
In some embodiments, positive or negative selection is accomplished
by incubating cells with one or more antibodies or other binding
agent that specifically bind to one or more surface markers
expressed or expressed (marker+) at a relatively higher level
(marker.sup.high) on the positively or negatively selected cells,
respectively.
[0645] In some embodiments, T cells are separated from a peripheral
blood mononuclear cell (PBMC) sample by negative selection of
markers expressed on non-T cells, such as B cells, monocytes, or
other white blood cells, such as CD14. In some aspects, a CD4+ or
CD8+ selection step is used to separate CD4+ helper and CD8+
cytotoxic T cells. Such CD4+ and CD8+ populations can be further
sorted into sub-populations by positive or negative selection for
markers expressed or expressed to a relatively higher degree on one
or more naive, memory, and/or effector T cell subpopulations.
[0646] In some embodiments, CD8+ cells are further enriched for or
depleted of naive, central memory, effector memory, and/or central
memory stem cells, such as by positive or negative selection based
on surface antigens associated with the respective subpopulation.
In some embodiments, enrichment for central memory T (TCM) cells is
carried out to increase efficacy, such as to improve long-term
survival, expansion, and/or engraftment following administration,
which in some aspects is particularly robust in such
sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et
al. (2012) J Immunother. 35(9):689-701. In some embodiments,
combining TCM-enriched CD8+ T cells and CD4+ T cells further
enhances efficacy.
[0647] In embodiments, memory T cells are present in both CD62L+
and CD62L- subsets of CD8+ peripheral blood lymphocytes. Peripheral
blood mononuclear cell (PBMC) can be enriched for or depleted of
CD62L-CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and
anti-CD62L antibodies.
[0648] In some embodiments, the enrichment for central memory T
(TCM) cells is based on positive or high surface expression of
CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it
is based on negative selection for cells expressing or highly
expressing CD45RA and/or granzyme B. In some aspects, isolation of
a CD8+ population enriched for TCM cells is carried out by
depletion of cells expressing CD4, CD14, CD45RA, and positive
selection or enrichment for cells expressing CD62L. In one aspect,
enrichment for central memory T (TCM) cells is carried out starting
with a negative fraction of cells selected based on CD4 expression,
which is subjected to a negative selection based on expression of
CD14 and CD45RA, and a positive selection based on CD62L. Such
selections in some aspects are carried out simultaneously and in
other aspects are carried out sequentially, in either order. In
some aspects, the same CD4 expression-based selection step used in
preparing the CD8+ cell population or subpopulation, also is used
to generate the CD4+ cell population or sub-population, such that
both the positive and negative fractions from the CD4-based
separation are retained and used in subsequent steps of the
methods, optionally following one or more further positive or
negative selection steps.
[0649] In a particular example, a sample of PBMCs or other white
blood cell sample is subjected to selection of CD4+ cells, where
both the negative and positive fractions are retained. The negative
fraction then is subjected to negative selection based on
expression of CD14 and CD45RA or ROR1, and positive selection based
on a marker characteristic of central memory T cells, such as CD62L
or CCR7, where the positive and negative selections are carried out
in either order.
[0650] CD4+ T helper cells are sorted into naive, central memory,
and effector cells by identifying cell populations that have cell
surface antigens. CD4+ lymphocytes can be obtained by standard
methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-,
CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory
CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector
CD4+ cells are CD62L- and CD45RO-.
[0651] In one example, to enrich for CD4+ cells by negative
selection, a monoclonal antibody cocktail typically includes
antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some
embodiments, the antibody or binding partner is bound to a solid
support or matrix, such as a magnetic bead or paramagnetic bead, to
allow for separation of cells for positive and/or negative
selection. For example, in some embodiments, the cells and cell
populations are separated or isolated using immune-magnetic (or
affinity-magnetic) separation techniques (reviewed in Methods in
Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2:
Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks
and U. Schumacher Humana Press Inc., Totowa, N.J.).
[0652] In some aspects, the sample or composition of cells to be
separated is incubated with small, magnetizable or magnetically
responsive material, such as magnetically responsive particles or
microparticles, such as paramagnetic beads (e.g., such as Dynabeads
or MACS beads). The magnetically responsive material, e.g.,
particle, generally is directly or indirectly attached to a binding
partner, e.g., an antibody, that specifically binds to a molecule,
e.g., surface marker, present on the cell, cells, or population of
cells that it is desired to separate, e.g., that it is desired to
negatively or positively select.
[0653] In some embodiments, the magnetic particle or bead comprises
a magnetically responsive material bound to a specific binding
member, such as an antibody or other binding partner. There are
many well-known magnetically responsive materials used in magnetic
separation methods. Suitable magnetic particles include those
described in Molday, U.S. Pat. No. 4,452,773, and in European
Patent Specification EP 452342 B, which are hereby incorporated by
reference. Colloidal sized particles, such as those described in
Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No.
5,200,084 are other examples.
[0654] The incubation generally is carried out under conditions
whereby the antibodies or binding partners, or molecules, such as
secondary antibodies or other reagents, which specifically bind to
such antibodies or binding partners, which are attached to the
magnetic particle or bead, specifically bind to cell surface
molecules if present on cells within the sample.
[0655] In some aspects, the sample is placed in a magnetic field,
and those cells having magnetically responsive or magnetizable
particles attached thereto will be attracted to the magnet and
separated from the unlabeled cells. For positive selection, cells
that are attracted to the magnet are retained; for negative
selection, cells that are not attracted (unlabeled cells) are
retained. In some aspects, a combination of positive and negative
selection is performed during the same selection step, where the
positive and negative fractions are retained and further processed
or subject to further separation steps.
[0656] In certain embodiments, the magnetically responsive
particles are coated in primary antibodies or other binding
partners, secondary antibodies, lectins, enzymes, or streptavidin.
In certain embodiments, the magnetic particles are attached to
cells via a coating of primary antibodies specific for one or more
markers. In certain embodiments, the cells, rather than the beads,
are labeled with a primary antibody or binding partner, and then
cell-type specific secondary antibody- or other binding partner
(e.g., streptavidin)-coated magnetic particles, are added. In
certain embodiments, streptavidin-coated magnetic particles are
used in conjunction with biotinylated primary or secondary
antibodies.
[0657] In some embodiments, the magnetically responsive particles
are left attached to the cells that are to be subsequently
incubated, cultured and/or engineered; in some aspects, the
particles are left attached to the cells for administration to a
patient. In some embodiments, the magnetizable or magnetically
responsive particles are removed from the cells. Methods for
removing magnetizable particles from cells are known and include,
e.g., the use of competing non-labeled antibodies, magnetizable
particles or antibodies conjugated to cleavable linkers, etc. In
some embodiments, the magnetizable particles are biodegradable.
[0658] In some embodiments, the affinity-based selection is via
magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn,
Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable
of high-purity selection of cells having magnetized particles
attached thereto. In certain embodiments, MACS operates in a mode
wherein the non-target and target species are sequentially eluted
after the application of the external magnetic field. That is, the
cells attached to magnetized particles are held in place while the
unattached species are eluted. Then, after this first elution step
is completed, the species that were trapped in the magnetic field
and were prevented from being eluted are freed in some manner such
that they can be eluted and recovered. In certain embodiments, the
non-target cells are labelled and depleted from the heterogeneous
population of cells.
[0659] In certain embodiments, the isolation or separation is
carried out using a system, device, or apparatus that carries out
one or more of the isolation, cell preparation, separation,
processing, incubation, culture, and/or formulation steps of the
methods. In some aspects, the system is used to carry out each of
these steps in a closed or sterile environment, for example, to
minimize error, user handling and/or contamination. In one example,
the system is a system as described in International Patent
Application, Publication Number WO2009/072003, or US 20110003380
A1.
[0660] In some embodiments, the system or apparatus carries out one
or more, e.g., all, of the isolation, processing, engineering, and
formulation steps in an integrated or self-contained system, and/or
in an automated or programmable fashion. In some aspects, the
system or apparatus includes a computer and/or computer program in
communication with the system or apparatus, which allows a user to
program, control, assess the outcome of, and/or adjust various
aspects of the processing, isolation, engineering, and formulation
steps.
[0661] In some aspects, the separation and/or other steps is
carried out using CliniMACS system (Miltenyi Biotec), for example,
for automated separation of cells on a clinical-scale level in a
closed and sterile system. Components can include an integrated
microcomputer, magnetic separation unit, peristaltic pump, and
various pinch valves. The integrated computer in some aspects
controls all components of the instrument and directs the system to
perform repeated procedures in a standardized sequence. The
magnetic separation unit in some aspects includes a movable
permanent magnet and a holder for the selection column. The
peristaltic pump controls the flow rate throughout the tubing set
and, together with the pinch valves, ensures the controlled flow of
buffer through the system and continual suspension of cells.
[0662] The CliniMACS system in some aspects uses antibody-coupled
magnetizable particles that are supplied in a sterile,
non-pyrogenic solution. In some embodiments, after labelling of
cells with magnetic particles the cells are washed to remove excess
particles. A cell preparation bag is then connected to the tubing
set, which in turn is connected to a bag containing buffer and a
cell collection bag. The tubing set consists of pre-assembled
sterile tubing, including a pre-column and a separation column, and
are for single use only. After initiation of the separation
program, the system automatically applies the cell sample onto the
separation column. Labeled cells are retained within the column,
while unlabeled cells are removed by a series of washing steps. In
some embodiments, the cell populations for use with the methods
described herein are unlabeled and are not retained in the column.
In some embodiments, the cell populations for use with the methods
described herein are labeled and are retained in the column. In
some embodiments, the cell populations for use with the methods
described herein are eluted from the column after removal of the
magnetic field, and are collected within the cell collection
bag.
[0663] In certain embodiments, separation and/or other steps are
carried out using the CliniMACS Prodigy system (Miltenyi Biotec).
The CliniMACS Prodigy system in some aspects is equipped with a
cell processing unity that permits automated washing and
fractionation of cells by centrifugation. The CliniMACS Prodigy
system can also include an onboard camera and image recognition
software that determines the optimal cell fractionation endpoint by
discerning the macroscopic layers of the source cell product. For
example, peripheral blood may be automatically separated into
erythrocytes, white blood cells and plasma layers. The CliniMACS
Prodigy system can also include an integrated cell cultivation
chamber which accomplishes cell culture protocols such as, e.g.,
cell differentiation and expansion, antigen loading, and long-term
cell culture. Input ports can allow for the sterile removal and
replenishment of media and cells can be monitored using an
integrated microscope. See, e.g., Klebanoff et al. (2012) J
Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82,
and Wang et al. (2012) J Immunother. 35(9):689-701.
[0664] In some embodiments, a cell population described herein is
collected and enriched (or depleted) via flow cytometry, in which
cells stained for multiple cell surface markers are carried in a
fluidic stream. In some embodiments, a cell population described
herein is collected and enriched (or depleted) via preparative
scale fluorescence activated cell sorting (FACS). In certain
embodiments, a cell population described herein is collected and
enriched (or depleted) by use of microelectromechanical systems
(MEMS) chips in combination with a FACS-based detection system
(see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10,
1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In
both cases, cells can be labeled with multiple markers, allowing
for the isolation of well-defined T cell subsets at high
purity.
[0665] In some embodiments, the antibodies or binding partners are
labeled with one or more detectable marker, to facilitate
separation for positive and/or negative selection. For example,
separation may be based on binding to fluorescently labeled
antibodies. In some examples, separation of cells based on binding
of antibodies or other binding partners specific for one or more
cell surface markers are carried in a fluidic stream, such as by
fluorescence-activated cell sorting (FACS), including preparative
scale (FACS) and/or microelectromechanical systems (MEMS) chips,
e.g., in combination with a flow-cytometric detection system. Such
methods allow for positive and negative selection based on multiple
markers simultaneously.
[0666] In some embodiments, the preparation methods include steps
for freezing, e.g., cryopreserving, the cells, either before or
after isolation, incubation, and/or engineering. In some
embodiments, the freeze and subsequent thaw step removes
granulocytes and, to some extent, monocytes in the cell population.
In some embodiments, the cells are suspended in a freezing
solution, e.g., following a washing step to remove plasma and
platelets. Any of a variety of known freezing solutions and
parameters in some aspects may be used. One example involves using
PBS containing 20% DMSO and 8% human serum albumin (HSA), or other
suitable cell freezing media. This can then be diluted 1:1 with
media so that the final concentration of DMSO and HSA are 10% and
4%, respectively. Other examples include Cryostor.RTM.,
CTL-Cryo.TM. ABC freezing media, and the like. The cells are then
frozen to -80 degrees C. at a rate of 1 degree per minute and
stored in the vapor phase of a liquid nitrogen storage tank.
[0667] In some embodiments, the provided methods include
cultivation, incubation, culture, and/or genetic engineering steps.
For example, in some embodiments, provided are methods for
incubating and/or engineering the depleted cell populations and
culture-initiating compositions.
[0668] Thus, in some embodiments, the cell populations are
incubated in a culture-initiating composition. The incubation
and/or engineering may be carried out in a culture vessel, such as
a unit, chamber, well, column, tube, tubing set, valve, vial,
culture dish, bag, or other container for culture or cultivating
cells.
[0669] In some embodiments, the cells are incubated and/or cultured
prior to or in connection with genetic engineering. The incubation
steps can include culture, cultivation, stimulation, activation,
and/or propagation. In some embodiments, the compositions or cells
are incubated in the presence of stimulating conditions or a
stimulatory agent. Such conditions include those designed to induce
proliferation, expansion, activation, and/or survival of cells in
the population, to mimic antigen exposure, and/or to prime the
cells for genetic engineering, such as for the introduction of a
recombinant antigen receptor.
[0670] The conditions can include one or more of particular media,
temperature, oxygen content, carbon dioxide content, time, agents,
e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory
factors, such as cytokines, chemokines, antigens, binding partners,
fusion proteins, recombinant soluble receptors, and any other
agents designed to activate the cells.
[0671] In some embodiments, the stimulating conditions or agents
include one or more agent, e.g., ligand, which is capable of
activating an intracellular signaling domain of a TCR complex. In
some aspects, the agent turns on or initiates TCR/CD3 intracellular
signaling cascade in a T cell. Such agents can include antibodies,
such as those specific for a TCR component and/or costimulatory
receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid
support such as a bead, and/or one or more cytokines. Optionally,
the expansion method may further comprise the step of adding
anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at
a concentration of at least about 0.5 ng/ml). In some embodiments,
the stimulating agents include IL-2 and/or IL-15, for example, an
IL-2 concentration of at least about 10 units/mL.
[0672] In some aspects, incubation is carried out in accordance
with techniques such as those described in U.S. Pat. No. 6,040,177
to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9):
651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al.
(2012) J Immunother. 35(9):689-701.
[0673] In some embodiments, the T cells are expanded by adding to
the culture-initiating composition feeder cells, such as
non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such
that the resulting population of cells contains at least about 5,
10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in
the initial population to be expanded); and incubating the culture
(e.g. for a time sufficient to expand the numbers of T cells). In
some aspects, the non-dividing feeder cells can comprise
gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC
are irradiated with gamma rays in the range of about 3000 to 3600
rads to prevent cell division. In some embodiments, the PBMC feeder
cells are inactivated with Mytomicin C. In some aspects, the feeder
cells are added to culture medium prior to the addition of the
populations of T cells.
[0674] In some embodiments, the stimulating conditions include
temperature suitable for the growth of human T lymphocytes, for
example, at least about 25 degrees Celsius, generally at least
about 30 degrees, and generally at or about 37 degrees Celsius.
Optionally, the incubation may further comprise adding non-dividing
EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can
be irradiated with gamma rays in the range of about 6000 to 10,000
rads. The LCL feeder cells in some aspects is provided in any
suitable amount, such as a ratio of LCL feeder cells to initial T
lymphocytes of at least about 10:1.
[0675] In embodiments, antigen-specific T cells, such as
antigen-specific CD4+ and/or CD8+ T cells, are obtained by
stimulating naive or antigen specific T lymphocytes with antigen.
For example, antigen-specific T cell lines or clones can be
generated to cytomegalovirus antigens by isolating T cells from
infected subjects and stimulating the cells in vitro with the same
antigen.
[0676] Assays
[0677] A variety of assays known in the art may be used to identify
and characterize an HLA-PEPTIDE ABP provided herein.
Binding, Competition, and Epitope Mapping Assays
[0678] Specific antigen-binding activity of an ABP provided herein
may be evaluated by any suitable method, including using SPR, BLI,
RIA and MSD-SET, as described elsewhere in this disclosure.
Additionally, antigen-binding activity may be evaluated by ELISA
assays, using flow cytometry, and/or Western blot assays.
[0679] Assays for measuring competition between two ABPs, or an ABP
and another molecule (e.g., one or more ligands of HLA-PEPTIDE such
as a TCR) are described elsewhere in this disclosure and, for
example, in Harlow and Lane, ABPs: A Laboratory Manual ch.14, 1988,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
incorporated by reference in its entirety.
[0680] Assays for mapping the epitopes to which an ABP provided
herein bind are described, for example, in Morris "Epitope Mapping
Protocols," in Methods in Molecular Biology vol. 66, 1996, Humana
Press, Totowa, N.J., incorporated by reference in its entirety. In
some embodiments, the epitope is determined by peptide competition.
In some embodiments, the epitope is determined by mass
spectrometry. In some embodiments, the epitope is determined by
mutagenesis. In some embodiments, the epitope is determined by
crystallography.
Assays for Effector Functions
[0681] Effector function following treatment with an ABP and/or
cell provided herein may be evaluated using a variety of in vitro
and in vivo assays known in the art, including those described in
Ravetch and Kinet, Annu. Rev. Immunol., 1991, 9:457-492; U.S. Pat.
Nos. 5,500,362, 5,821,337; Hellstrom et al., Proc. Nat'l Acad. Sci.
USA, 1986, 83:7059-7063; Hellstrom et al., Proc. Nat'l Acad. Sci.
USA, 1985, 82:1499-1502; Bruggemann et al., J Exp. Med., 1987,
166:1351-1361; Clynes et al., Proc. Nat'l Acad. Sci. USA, 1998,
95:652-656; WO 2006/029879; WO 2005/100402; Gazzano-Santoro et al.,
J. Immunol. Methods, 1996, 202:163-171; Cragg et al., Blood, 2003,
101:1045-1052; Cragg et al. Blood, 2004, 103:2738-2743; and Petkova
et al., Int'l. Immunol., 2006, 18:1759-1769; each of which is
incorporated by reference in its entirety.
[0682] Pharmaceutical Compositions
[0683] An ABP, cell, or HLA-PEPTIDE target provided herein can be
formulated in any appropriate pharmaceutical composition and
administered by any suitable route of administration. Suitable
routes of administration include, but are not limited to, the
intra-arterial, intradermal, intramuscular, intraperitoneal,
intravenous, nasal, parenteral, pulmonary, and subcutaneous
routes.
[0684] The pharmaceutical composition may comprise one or more
pharmaceutical excipients. Any suitable pharmaceutical excipient
may be used, and one of ordinary skill in the art is capable of
selecting suitable pharmaceutical excipients. Accordingly, the
pharmaceutical excipients provided below are intended to be
illustrative, and not limiting. Additional pharmaceutical
excipients include, for example, those described in the Handbook of
Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009),
incorporated by reference in its entirety.
[0685] In some embodiments, the pharmaceutical composition
comprises an anti-foaming agent. Any suitable anti-foaming agent
may be used. In some aspects, the anti-foaming agent is selected
from an alcohol, an ether, an oil, a wax, a silicone, a surfactant,
and combinations thereof. In some aspects, the anti-foaming agent
is selected from a mineral oil, a vegetable oil, ethylene bis
stearamide, a paraffin wax, an ester wax, a fatty alcohol wax, a
long chain fatty alcohol, a fatty acid soap, a fatty acid ester, a
silicon glycol, a fluorosilicone, a polyethylene
glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon
dioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate,
ethyl alcohol, 2-ethyl-hexanol, dimethicone, oleyl alcohol,
simethicone, and combinations thereof.
[0686] In some embodiments, the pharmaceutical composition
comprises a co-solvent. Illustrative examples of co-solvents
include ethanol, poly(ethylene) glycol, butylene glycol,
dimethylacetamide, glycerin, propylene glycol, and combinations
thereof.
[0687] In some embodiments, the pharmaceutical composition
comprises a buffer. Illustrative examples of buffers include
acetate, borate, carbonate, lactate, malate, phosphate, citrate,
hydroxide, diethanolamine, monoethanolamine, glycine, methionine,
guar gum, monosodium glutamate, and combinations thereof.
[0688] In some embodiments, the pharmaceutical composition
comprises a carrier or filler. Illustrative examples of carriers or
fillers include lactose, maltodextrin, mannitol, sorbitol,
chitosan, stearic acid, xanthan gum, guar gum, and combinations
thereof.
[0689] In some embodiments, the pharmaceutical composition
comprises a surfactant. Illustrative examples of surfactants
include d-alpha tocopherol, benzalkonium chloride, benzethonium
chloride, cetrimide, cetylpyridinium chloride, docusate sodium,
glyceryl behenate, glyceryl monooleate, lauric acid, macrogol 15
hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene
alkyl ethers, polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene stearates, polyoxylglycerides, sodium lauryl
sulfate, sorbitan esters, vitamin E polyethylene(glycol) succinate,
and combinations thereof.
[0690] In some embodiments, the pharmaceutical composition
comprises an anti-caking agent. Illustrative examples of
anti-caking agents include calcium phosphate (tribasic),
hydroxymethyl cellulose, hydroxypropyl cellulose, magnesium oxide,
and combinations thereof.
[0691] Other excipients that may be used with the pharmaceutical
compositions include, for example, albumin, antioxidants,
antibacterial agents, antifungal agents, bioabsorbable polymers,
chelating agents, controlled release agents, diluents, dispersing
agents, dissolution enhancers, emulsifying agents, gelling agents,
ointment bases, penetration enhancers, preservatives, solubilizing
agents, solvents, stabilizing agents, sugars, and combinations
thereof. Specific examples of each of these agents are described,
for example, in the Handbook of Pharmaceutical Excipients, Rowe et
al. (Eds.) 6th Ed. (2009), The Pharmaceutical Press, incorporated
by reference in its entirety.
[0692] In some embodiments, the pharmaceutical composition
comprises a solvent. In some aspects, the solvent is saline
solution, such as a sterile isotonic saline solution or dextrose
solution. In some aspects, the solvent is water for injection.
[0693] In some embodiments, the pharmaceutical compositions are in
a particulate form, such as a microparticle or a nanoparticle.
Microparticles and nanoparticles may be formed from any suitable
material, such as a polymer or a lipid. In some aspects, the
microparticles or nanoparticles are micelles, liposomes, or
polymersomes.
[0694] Further provided herein are anhydrous pharmaceutical
compositions and dosage forms comprising an ABP, since water can
facilitate the degradation of some ABPs.
[0695] Anhydrous pharmaceutical compositions and dosage forms
provided herein can be prepared using anhydrous or low moisture
containing ingredients and low moisture or low humidity conditions.
Pharmaceutical compositions and dosage forms that comprise lactose
and at least one active ingredient that comprises a primary or
secondary amine can be anhydrous if substantial contact with
moisture and/or humidity during manufacturing, packaging, and/or
storage is expected.
[0696] An anhydrous pharmaceutical composition should be prepared
and stored such that its anhydrous nature is maintained.
Accordingly, anhydrous compositions can be packaged using materials
known to prevent exposure to water such that they can be included
in suitable formulary kits. Examples of suitable packaging include,
but are not limited to, hermetically sealed foils, plastics, unit
dose containers (e.g., vials), blister packs, and strip packs.
[0697] In certain embodiments, an ABP and/or cell provided herein
is formulated as parenteral dosage forms. Parenteral dosage forms
can be administered to subjects by various routes including, but
not limited to, subcutaneous, intravenous (including infusions and
bolus injections), intramuscular, and intra-arterial. Because their
administration typically bypasses subjects' natural defenses
against contaminants, parenteral dosage forms are typically,
sterile or capable of being sterilized prior to administration to a
subject. Examples of parenteral dosage forms include, but are not
limited to, solutions ready for injection, dry (e.g., lyophilized)
products ready to be dissolved or suspended in a pharmaceutically
acceptable vehicle for injection, suspensions ready for injection,
and emulsions.
[0698] Suitable vehicles that can be used to provide parenteral
dosage forms are well known to those skilled in the art. Examples
include, but are not limited to: Water for Injection USP; aqueous
vehicles such as, but not limited to, Sodium Chloride Injection,
Ringer's Injection, Dextrose Injection, Dextrose and Sodium
Chloride Injection, and Lactated Ringer's Injection; water miscible
vehicles such as, but not limited to, ethyl alcohol, polyethylene
glycol, and polypropylene glycol; and non-aqueous vehicles such as,
but not limited to, corn oil, cottonseed oil, peanut oil, sesame
oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
[0699] Excipients that increase the solubility of one or more of
the ABPs and/or cells disclosed herein can also be incorporated
into the parenteral dosage forms.
[0700] In some embodiments, the parenteral dosage form is
lyophilized. Exemplary lyophilized formulations are described, for
example, in U.S. Pat. Nos. 6,267,958 and 6,171,586; and WO
2006/044908; each of which is incorporated by reference in its
entirety.
[0701] In human therapeutics, the doctor will determine the
posology which he considers most appropriate according to a
preventive or curative treatment and according to the age, weight,
condition and other factors specific to the subject to be
treated.
[0702] In certain embodiments, a composition provided herein is a
pharmaceutical composition or a single unit dosage form.
Pharmaceutical compositions and single unit dosage forms provided
herein comprise a prophylactically or therapeutically effective
amount of one or more prophylactic or therapeutic ABP.
[0703] The amount of the ABP, cell, or composition which will be
effective in the prevention or treatment of a disorder or one or
more symptoms thereof will vary with the nature and severity of the
disease or condition, and the route by which the ABP and/or cell is
administered. The frequency and dosage will also vary according to
factors specific for each subject depending on the specific therapy
(e.g., therapeutic or prophylactic agents) administered, the
severity of the disorder, disease, or condition, the route of
administration, as well as age, body, weight, response, and the
past medical history of the subject. Effective doses may be
extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0704] Different therapeutically effective amounts may be
applicable for different diseases and conditions, as will be
readily known by those of ordinary skill in the art. Similarly,
amounts sufficient to prevent, manage, treat or ameliorate such
disorders, but insufficient to cause, or sufficient to reduce,
adverse effects associated with the ABPs and/or cells provided
herein are also encompassed by the dosage amounts and dose
frequency schedules provided herein. Further, when a subject is
administered multiple dosages of a composition provided herein, not
all of the dosages need be the same. For example, the dosage
administered to the subject may be increased to improve the
prophylactic or therapeutic effect of the composition or it may be
decreased to reduce one or more side effects that a particular
subject is experiencing.
[0705] In certain embodiments, treatment or prevention can be
initiated with one or more loading doses of an ABP or composition
provided herein followed by one or more maintenance doses.
[0706] In certain embodiments, a dose of an ABP, cell, or
composition provided herein can be administered to achieve a
steady-state concentration of the ABP and/or cell in blood or serum
of the subject. The steady-state concentration can be determined by
measurement according to techniques available to those of skill or
can be based on the physical characteristics of the subject such as
height, weight and age.
[0707] As discussed in more detail elsewhere in this disclosure, an
ABP and/or cell provided herein may optionally be administered with
one or more additional agents useful to prevent or treat a disease
or disorder. The effective amount of such additional agents may
depend on the amount of ABP present in the formulation, the type of
disorder or treatment, and the other factors known in the art or
described herein.
[0708] Therapeutic Applications
[0709] For therapeutic applications, ABPs and/or cells are
administered to a mammal, generally a human, in a pharmaceutically
acceptable dosage form such as those known in the art and those
discussed above. For example, ABPs and/or cells may be administered
to a human intravenously as a bolus or by continuous infusion over
a period of time, by intramuscular, intraperitoneal,
intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, or intratumoral routes. The ABPs also are suitably
administered by peritumoral, intralesional, or perilesional routes,
to exert local as well as systemic therapeutic effects. The
intraperitoneal route may be particularly useful, for example, in
the treatment of ovarian tumors.
[0710] The ABPs and/or cells provided herein can be useful for the
treatment of any disease or condition involving HLA-PEPTIDE. In
some embodiments, the disease or condition is a disease or
condition that can benefit from treatment with an anti-HLA-PEPTIDE
ABP and/or cell. In some embodiments, the disease or condition is a
tumor. In some embodiments, the disease or condition is a cell
proliferative disorder. In some embodiments, the disease or
condition is a cancer.
[0711] In some embodiments, the ABPs and/or cells provided herein
are provided for use as a medicament. In some embodiments, the ABPs
and/or cells provided herein are provided for use in the
manufacture or preparation of a medicament. In some embodiments,
the medicament is for the treatment of a disease or condition that
can benefit from an anti-HLA-PEPTIDE ABP and/or cell. In some
embodiments, the disease or condition is a tumor. In some
embodiments, the disease or condition is a cell proliferative
disorder. In some embodiments, the disease or condition is a
cancer.
[0712] In some embodiments, provided herein is a method of treating
a disease or condition in a subject in need thereof by
administering an effective amount of an ABP and/or cell provided
herein to the subject. In some aspects, the disease or condition is
a cancer.
[0713] In some embodiments, provided herein is a method of treating
a disease or condition in a subject in need thereof by
administering an effective amount of an ABP and/or cell provided
herein to the subject, wherein the disease or condition is a
cancer, and the cancer is selected from a solid tumor and a
hematological tumor.
[0714] In some embodiments, provided herein is a method of
modulating an immune response in a subject in need thereof,
comprising administering to the subject an effective amount of an
ABP and/or cell or a pharmaceutical composition disclosed
herein.
[0715] Combination Therapies
[0716] In some embodiments, an ABP and/or cell provided herein is
administered with at least one additional therapeutic agent. Any
suitable additional therapeutic agent may be administered with an
ABP and/or cell provided herein. An additional therapeutic agent
can be fused to an ABP. In some aspects, the additional therapeutic
agent is selected from radiation, a cytotoxic agent, a toxin, a
chemotherapeutic agent, a cytostatic agent, an anti-hormonal agent,
an EGFR inhibitor, an immunomodulatory agent, an anti-angiogenic
agent, and combinations thereof. In some embodiments, the
additional therapeutic agent is an ABP.
[0717] Diagnostic Methods
[0718] Also provided are methods for predicting and/or detecting
the presence of a given HLA-PEPTIDE on a cell from a subject. Such
methods may be used, for example, to predict and evaluate
responsiveness to treatment with an ABP and/or cell provided
herein.
[0719] In some embodiments, a blood or tumor sample is obtained
from a subject and the fraction of cells expressing HLA-PEPTIDE is
determined. In some aspects, the relative amount of HLA-PEPTIDE
expressed by such cells is determined. The fraction of cells
expressing HLA-PEPTIDE and the relative amount of HLA-PEPTIDE
expressed by such cells can be determined by any suitable method.
In some embodiments, flow cytometry is used to make such
measurements. In some embodiments, fluorescence assisted cell
sorting (FACS) is used to make such measurement. See Li et al., J.
Autoimmunity, 2003, 21:83-92 for methods of evaluating expression
of HLA-PEPTIDE in peripheral blood.
[0720] In some embodiments, detecting the presence of a given
HLA-PEPTIDE on a cell from a subject is performed using
immunoprecipitation and mass spectrometry. This can be performed by
obtaining a tumor sample (e.g., a frozen tumor sample) such as a
primary tumor specimen and applying immunoprecipitation to isolate
one or more peptides. The HLA alleles of the tumor sample can be
determined experimentally or obtained from a third party source.
The one or more peptides can be subjected to mass spectrometry (MS)
to determine their sequence(s). The spectra from the MS can then be
searched against a database. An example is provided in the Examples
section below.
[0721] In some embodiments, predicting the presence of a given
HLA-PEPTIDE on a cell from a subject is performed using a
computer-based model applied to the peptide sequence and/or RNA
measurements of one or more genes comprising that peptide sequence
(e.g., RNA seq or RT-PCR, or nanostring) from a tumor sample. The
model used can be as described in international patent application
no. PCT/US2016/067159, herein incorporated by reference, in its
entirety, for all purposes.
[0722] Kits
[0723] Also provided are kits comprising an ABP and/or cell
provided herein. The kits may be used for the treatment,
prevention, and/or diagnosis of a disease or disorder, as described
herein.
[0724] In some embodiments, the kit comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
and IV solution bags. The containers may be formed from a variety
of materials, such as glass or plastic. The container holds a
composition that is by itself, or when combined with another
composition, effective for treating, preventing and/or diagnosing a
disease or disorder. The container may have a sterile access port.
For example, if the container is an intravenous solution bag or a
vial, it may have a port that can be pierced by a needle. At least
one active agent in the composition is an ABP provided herein. The
label or package insert indicates that the composition is used for
treating the selected condition.
[0725] In some embodiments, the kit comprises (a) a first container
with a first composition contained therein, wherein the first
composition comprises an ABP and/or cell provided herein; and (b) a
second container with a second composition contained therein,
wherein the second composition comprises a further therapeutic
agent. The kit in this embodiment can further comprise a package
insert indicating that the compositions can be used to treat a
particular condition, e.g., cancer.
[0726] Alternatively, or additionally, the kit may further comprise
a second (or third) container comprising a
pharmaceutically-acceptable excipient. In some aspects, the
excipient is a buffer. The kit may further include other materials
desirable from a commercial and user standpoint, including filters,
needles, and syringes.
EXAMPLES
[0727] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures,
etc.), but some experimental error and deviation should, of course,
be allowed for.
[0728] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of protein chemistry,
biochemistry, recombinant DNA techniques and pharmacology, within
the skill of the art. Such techniques are explained fully in the
literature. See, e.g., T. E. Creighton, Proteins: Structures and
Molecular Properties (W.H. Freeman and Company, 1993); A. L.
Lehninger, Biochemistry (Worth Publishers, Inc., current addition);
Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd
Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan
eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences,
18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey
and Sundberg Advanced Organic Chemistry 3.sup.rd Ed. (Plenum Press)
Vols A and B (1992).
Example 1: Identification of Predicted HLA-PEPTIDE Complexes (Table
A)
[0729] We identified two classes of cancer specific HLA-peptide
targets: The first class (cancer testis antigens, CTAs) are not
expressed or are expressed at minimal levels in most normal tissues
and expressed in tumor samples. The second class (tumor associated
antigens, TAAs) are expressed highly in tumor samples and may have
low expression in normal tissues.
[0730] We identified gene targets using three computational steps:
First, we identified genes with low or no expression in most normal
tissues using data available through the Genotype-Tissue Expression
(GTEx) Project [1]. We obtained aggregated gene expression data
from the Genotype-Tissue Expression (GTEx) Project (version V7p2).
This dataset comprised 11,688 post-mortem samples from 714
individuals and fifty-three different tissue types. Expression was
measured using RNA-Seq and computationally processed according to
the GTEx standard pipeline
(https://www.gtexportal.org/home/documentationPage). Gene
expression was calculated using the sum of isoform expression that
were calculated using RSEM v1.2.22 [2].
[0731] Next, we identified which of those genes are aberrantly
expressed in cancer samples using data from The Cancer Genome Atlas
(TCGA) Research Network: http://cancergenome.nih.gov/. We examined
11,093 samples available from TCGA (Data Release 6.0). Because GTEx
and TCGA use different annotations of the human genome in their
computational analyses, we only included genes for which there were
available ENCODE mappings between the two datasets.
[0732] Finally, in these genes, we identified which peptides are
likely to be presented as cell surface antigens by MHC Class I
proteins using a deep learning model trained on HLA presented
peptides sequenced by tandem mass spectrometry (MS/MS), as
described in international patent application no.
PCT/US2016/067159, herein incorporated by reference, in its
entirety, for all purposes.
[0733] Specific criteria for the two classes of genes is given
below.
[0734] CTA Inclusion Criteria
[0735] To identify the CTAs, we sought to define a criteria to
exclude genes that were expressed in normal tissue that was strict
enough to ensure tumor specificity, but would not exclude non-zero
measurements arising from potential artifacts such as read
misalignment. Genes were eligible for inclusion as CTAs if they met
the following criteria: The median GTEx expression in each organ
that was a part of the brain, heart, or lung was less than 0.1
transcripts per million (TPM) with no one sample exceeding 5 TPM.
The median GTEx expression in other essential organs was less than
2 TPM with no one sample exceeding 10 TPM. Expression was ignored
for organs classified as non-essential (testis, thyroid, and minor
salivary gland). Genes were considered expressed in tumor samples
if they had expression in TCGA of greater than 20 TPM in at least
30 samples.
[0736] We then examined the distribution of the expression of the
remaining genes across the TCGA samples. When we examined the known
CTAs, e.g. the MAGE family of genes, we observed that the
expression these genes in log space was generally characterized by
a bimodal distribution. This distribution consisted of a left mode
around a lower expression value and a right mode (or thick tail) at
a higher expression level. This expression pattern is consistent
with a biological model in which some minimal expression is
detected at baseline in all samples and higher expression of the
gene is observed in a subset of tumors experiencing epigenetic
dysregulation. We reviewed the distribution of expression of each
gene across TCGA samples and discarded those where we observed only
a unimodal distribution with no significant right-hand tail.
[0737] TAA Inclusion Criteria
[0738] The TAAs were identified by focusing on genes with much
higher expression in tumor tissues than in normal tissue: We first
identified genes with a median TPM of less than 10 in all GTEx
essential, normal tissues and then selected the subset which had
expression of greater than 100 TPM in at least one TCGA tumor
tissues. Then, we examined the distribution of each of these genes
and selected those with a bimodal distribution of expression, as
well as additional evidence of significantly elevated expression in
one or more tumor types.
[0739] Lists were further reviewed to eliminate genes which are
known to have expression in tissues not adequately represented in
GTEx or which could have originated from immune cell infiltrates
within the tumor. These steps left of us with a final list of 56
CTA and 58 TAA genes.
[0740] We also added peptides from two additional proteins known to
be present in cancer. We added the junction peptides from the
EGFR-SEPT14 fusion protein [3] and we added peptides from KLK3
(PSA). We also added peptides from two genes from the same gene
family as PSA: KLK2 and KLK4.
[0741] To identify the peptides that are likely to be presented as
cell surface antigens by MHC Class I proteins, we used a sliding
window to parse each of these proteins into its constituent 8-11
amino acid sequences. We processed these peptides and their
flanking sequences with the HLA peptide presentation deep learning
model to calculate the likelihood of presentation of each peptide
at the max expression level observed for this gene in TCGA. We
considered a peptide likely to be presented (i.e., a candidate
target) if its quantile normalized probability of presentation
calculated by our model was greater than 0.001.
[0742] The results are shown in Table A. This table is included in
PCT/US2018/06793, filed on Dec. 28, 2018, which is incorporated by
reference in its entirety.
[0743] In summary, the example provides a large set of
tumor-specific HLA-PEPTIDEs that can be pursued as candidate
targets for ABP development.
REFERENCES
[0744] 1. Consortium, G. T., The Genotype-Tissue Expression (GTEx)
project. Nat Genet, 2013. 45(6): p. 580-5. [0745] 2. Li B, Dewey C
N., RSEM: accurate transcript quantificationfrom RNA-Seq data with
or without a reference genome. BMC Bioinformatics. 2011 Aug. 4;
12:323. [0746] 3. Frattini V, Trifonov V, Chan J M, Castano A, Lia
M, Abate F, Keir S T, Ji A X, Zoppoli P, Niola F, Danussi C,
Dolgalev I, Porrati P, Pellegatta S, Heguy A, Gupta G, Pisapia D J,
Canoll P, Bruce I N, McLendon R E, Yan H, Aldape K, Finocchiaro G,
Mikkelsen T, Prive G G, Bigner D D, Lasorella A, Rabadan R,
Iavarone A. The integrated landscape of driver genomic alterations
in glioblastoma. Nat Genet. 2013 October; 45(10):1141-9.
Example 2: Validation of Predicted HLA-PEPTIDE Complexes
[0747] The presence of peptides from the HLA-PEPTIDE complexes of
Tables A, A1, and A2 was determined using mass spectrometry (MS) on
tumor samples known to be positive for each given HLA allele from
the respective HLA-PEPTIDE complex.
[0748] Isolation of HLA-peptide molecules was performed using
classic immunoprecipitation (IP) methods after lysis and
solubilization of the tissue sample (1-4). Fresh frozen tissue was
first frozen in liquid nitrogen and pulverized (CryoPrep; Covaris,
Woburn, Mass.). One tenth of the sample was aliquoted for genomic
sequencing efforts and lysis buffer (1% CHAPS, 20 mM Tris-HCl, 150
mM NaCl, protease and phosphatase inhibitors, pH=8) was added to
solubilize the remaining pulverized tissue. The sample lysate was
spun at 4.degree. C. for 2 hrs to pellet debris. The clarified
lysate was used for the HLA specific IP.
[0749] Immunoprecipitation was performed using antibodies coupled
to beads where the antibody was specific for HLA molecules. For a
pan-Class I HLA immunoprecipitation, the antibody W6/32 (5) was
used, for Class II HLA-DR, antibody L243 (6) was used. Antibody was
covalently attached to NHS-sepharose beads during overnight
incubation. After covalent attachment, the beads were washed and
aliquoted for IP. Additional methods for IP can be used including
but not limited to Protein A/G capture of antibody, magnetic bead
isolation, or other methods commonly used for
immunoprecipitation.
[0750] The lysate was added to the antibody beads and rotated at
4.degree. C. overnight for the immunoprecipitation. After
immunoprecipitation, the beads were removed from the lysate and the
lysate was stored for additional experiments, including additional
IPs. The TP beads were washed to remove non-specific binding and
the HLA/peptide complex was eluted from the beads with 2N acetic
acid. The protein components were removed from the peptides using a
molecular weight spin column or C18 cleanup step. The resultant
peptides were taken to dryness by SpeedVac evaporation and can be
stored at -20.degree. C. prior to MS analysis.
[0751] Dried peptides were reconstituted in HPLC buffer A and
loaded onto a C-18 microcapillary HPLC column for gradient elution
in to the mass spectrometer. A gradient of 0-40% B (solvent A--0.1%
formic acid, solvent B--0.1% formic acid in 80% acetonitrile) in
180 minutes was used to elute the peptides into the Fusion Lumos
mass spectrometer (Thermo). MS1 spectra of peptide mass/charge
(m/z) were collected in the Orbitrap detector with 120,000
resolution followed by 20 MS2 scans. Selection of MS2 ions was
performed using data dependent acquisition mode and dynamic
exclusion of 30 sec after MS2 selection of an ion. Automatic gain
control (AGC) for MS1 scans was set to 4.times.105 and for MS2
scans was set to 1.times.104. For sequencing HLA peptides, +1, +2
and +3 charge states can be selected for MS2 fragmentation.
Alternatively, MS2 spectra can be acquired using mass targeting
methods where only masses listed in the inclusion list were
selected for isolation and fragmentation. This was commonly
referred to as Targeted Mass Spectrometry and was performed in
either a qualitative manner or can be quantitative. Quantitation
methods require each peptide to be quantitated to be synthesized
using heavy labeled amino acids. (Doerr 2013)
[0752] MS2 spectra from each analysis were searched against a
protein database using Comet (7-8) and the peptide identification
was scored using Percolator (9-11) or using the integrated de novo
sequencing and database search algorithm of PEAKS. Peptides from
targeted MS2 experiments were analyzed using Skyline (Lindsay K.
Pino et al. 2017) or other method to analyze predicted fragment
ions.
[0753] The presence of multiple peptides from the predicted
HLA-PEPTIDE complexes was determined using mass spectrometry (MS)
on various tumor samples known to be positive for each given HLA
allele from the respective HLA-PEPTIDE complex.
[0754] Representative spectra data for selected HLA-restricted
peptides is shown in FIGS. 51-63. Each spectra contains the peptide
fragmentation information as well as information related to the
patient sample, including HLA types.
[0755] The spontaneous modification of amino acids can occur to
many amino acids. Cysteine was especially susceptible to this
modification and can be oxidized or modified with a free cysteine.
Additionally N-terminal glutamine amino acids can be converted to
pyro-glutamic acid. Since each of these modifications results in a
change in mass, they can be definitively assigned in the MS2
spectra. To use these peptides in preparation of ABPs the peptide
may need to contain the same modification as seen in the mass
spectrometer. These modifications can be created using simple
laboratory and peptide synthesis methods (Lee et al.; Ref 14).
REFERENCES
[0756] (1) Hunt D F, Henderson R A, Shabanowitz J, Sakaguchi K,
Michel H, Sevilir N, Cox A L, Appella E, Engelhard V H.
Characterization of peptides bound to the class I MHC molecule
HLA-A2.1 by mass spectrometry. Science 1992. 255: 1261-1263. [0757]
(2) Zarling A L, Polefrone J M, Evans A M, Mikesh L M, Shabanowitz
J, Lewis S T, Engelhard V H, Hunt D F. Identification of class I
MHC-associated phosphopeptides as targets for cancer
immunotherapy._Proc Natl Acad Sci USA. 2006 Oct. 3;
103(40):14889-94. [0758] (3) Bassani-Sternberg M,
Pletscher-Frankild S, Jensen L J, Mann M. Mass spectrometry of
human leukocyte antigen class I peptidomes reveals strong effects
of protein abundance and turnover on antigen presentation. Mol Cell
Proteomics. 2015 March; 14(3):658-73. doi: 10.1074/mcp.M114.042812.
[0759] (4) Abelin J G, Trantham P D, Penny S A, Patterson A M, Ward
S T, Hildebrand W H, Cobbold M, Bai D L, Shabanowitz J, Hunt D F.
Complementary IMAC enrichment methods for HLA-associated
phosphopeptide identification by mass spectrometry. Nat Protoc.
2015 September; 10(9):1308-18. doi: 10.1038/nprot.2015.086. Epub
2015 Aug. 6 [0760] (5) Barnstable C J, Bodmer W F, Brown G, Galfre
G, Milstein C, Williams A F, Ziegler A. Production of monoclonal
antibodies to group A erythrocytes, HLA and other human cell
surface antigens-new tools for genetic analysis. Cell. 1978 May;
14(1):9-20. [0761] (6) Goldman J M, Hibbin J, Kearney L, Orchard K,
Th'ng K H. HLA-D R monoclonal antibodies inhibit the proliferation
of normal and chronic granulocytic leukaemia myeloid progenitor
cells. Br J Haematol. 1982 November; 52(3):411-20. [0762] (7) Eng J
K, Jahan T A, Hoopmann M R. Comet: an open-source M S/M S sequence
database search tool. Proteomics. 2013 January; 13(1):22-4. doi:
10.1002/pmic.201200439. Epub 2012 Dec. 4. [0763] (8) Eng J K,
Hoopmann M R, Jahan T A, Egertson J D, Noble W S, MacCoss M J. A
deeper look into Comet--implementation and features. J Am Soc Mass
Spectrom. 2015 November; 26(11):1865-74. doi:
10.1007/s13361-015-1179-x. Epub 2015 Jun. 27. [0764] (9) Lukas
Kall, Jesse Canterbury, Jason Weston, William Stafford Noble and
Michael J. MacCoss. Semi-supervised learning for peptide
identification from shotgun proteomics datasets. Nature Methods
4:923-925, November 2007 [0765] (10) Lukas Kall, John D. Storey,
Michael J. MacCoss and William Stafford Noble. Assigning confidence
measures to peptides identified by tandem mass spectrometry.
Journal of Proteome Research, 7(1):29-34, January 2008 [0766] (11)
Lukas Kall, John D. Storey and William Stafford Noble.
Nonparametric estimation of posterior error probabilities
associated with peptides identified by tandem mass spectrometry.
Bioinformatics, 24(16):i42-i48, August 2008 [0767] (12) Doerr, A.
(2013) Mass Spectrometry-based targeted proteomics. Nature Methods,
10, 23. [0768] (13) Lindsay K. Pino, Brian C. Searle, James G.
Bollinger, Brook Nunn, Brendan MacLean & M. J. MacCoss (2017)
The Skyline ecosystem: Informatics for quantitative mass
spectrometry proteomics. Mass Spectrometry Reviews. [0769] (14) Lee
W Thompson; Kevin T Hogan; Jennifer A Caldwell; Richard A Pierce;
Ronald C Hendrickson; Donna H Deacon; Robert E Settlage; Laurence H
Brinckerhoff, Victor H Engelhard; Jeffrey Shabanowitz; Donald F
Hunt; Craig L Slingluff. Preventing the spontaneous modification of
an HLA-A2-restricted peptide at an N-terminal glutamine or an
internal cysteine residue enhances peptide antigenicity. Journal of
Immunotherapy (Hagerstown, Md.: 1997). 27(3):177-83, MAY 2004.
Example 3: Identification of Antibodies and Antigen Binding
Fragments Thereof that Bind HLA-PEPTIDE Targets HLA-B*35:01
EVDPIGHVY, HLA-A*02:01 AIFPGAVPAA, and HLA-A*01:01 ASSLPTTMNY
[0770] Overview
[0771] The following exemplification demonstrates that antibodies
(Abs) can be identified that recognize tumor-specific
HLA-restricted peptides. The overall epitope that is recognized by
such Abs generally comprises a composite surface of both the
peptide as well as the HLA protein presenting that particular
peptide. Abs that recognize HLA complexes in a peptide-specific
manner are often referred to as T cell receptor (TCR)-like Abs or
TCR-mimetic Abs. The HLA-PEPTIDE target antigens that were selected
for antibody discovery, derived from the tumor-specific gene
product MAGEA6, FOXE1, MAGE3/6, were HLA-B*35:01_EVDPIGHVY
(HLA-PEPTIDE target "G5"), HLA-A*02:01_AIFPGAVPAA (HLA-PEPTIDE
target "G8"), and HLA-A*01:01_ASSLPTTMNY (HLA-PEPTIDE target
"G10"), respectively. Cell surface presentation of these
HLA-PEPTIDE targets was confirmed by mass spectrometry analysis of
HLA complexes obtained from tumor samples as described in Example
2. Representative plots are depicted in FIGS. 25-27.
[0772] HLA-PEPTIDE Target Complexes and Counterscreen Peptide-HLA
Complexes
[0773] The HLA-PEPTIDE targets G5, G8, G10, as well as
counterscreen negative control peptide-HLAs, were produced
recombinantly using conditional ligands for HLA molecules using
established methods. In all, 18 counterscreen HLA-peptides were
generated for each of the HLA-PEPTIDE targets. The 18 counterscreen
HLA-peptides were designed such that (A) the negative control
peptide was known to be presented by the same HLA subtype (i.e. the
HLA-related controls) or (B) the negative control peptides were
known to be presented by a different HLA subtype. The grouping of
the target and the negative control peptide-HLA complexes for
screen 1 is shown in FIG. 3 (with detailed sequence information
provided in Table 1), and for screen 2 shown in FIG. 4 (with
detailed sequence information provided in Table 2.
TABLE-US-00033 TABLE 1 HLA-PEPTIDE sequence design for Screen 1
negative control peptides and ''G5'' target Group HLA Peptide Gene
Target G1 HLA-A*02:01 LLFGYPVYV Neg Ctrl 1 HLA-A*02:01 GILGFVFTL
Neg Ctrl 2 HLA-A*02:01 FLLTRILTI Neg Ctrl 3 G2 HLA-A*01:01
YSEHPTFTSQY Neg Ctrl 1 HLA-A*01:01 VSDGGPNLY Neg Ctrl 2 HLA-A*01:01
ATDALMTGY Neg Ctrl 3 G3 HLA-A*11:01 IVTDFSVIK Neg Ctrl 1
HLA-A*11:01 KSMREEYRK Neg Ctrl 2 HLA-A*11:01 SSCSSCPLSK Neg Ctrl 3
G4 HLA-A*11:01 ATIGTAMYK Neg Ctrl 1 HLA-A*11:01 AVFDRKSDAK Neg Ctrl
2 HLA-A*11:01 SIIPSGPLK Neg Ctrl 3 G5 HLA-B*35:01 EVDPIGHVY MAGEA6
Target HLA-B*35:01 IPSINVHHY Neg Ctrl 1 HLA-B*35:01 EPLPQGQLTAY Neg
Ctrl 2 HLA-B*35:01 VPLDEDFRKY Neg Ctrl 3 G6 HLA-A*03:01 RLRAEAQVK
Neg Ctrl 1 HLA-A*03:01 RLRPGGKKK Neg Ctrl 2 HLA-A*03:01 QVPLRPMTYK
Neg Ctrl 3
TABLE-US-00034 TABLE 2 HLA-PEPTIDE sequence design for Screen 2
negative control peptides, G8, and G10 targets* Group HLA Peptide
Gene Target G7/G8.dagger-dbl. A*02:01 LLFGYPVYV Neg Ctrl 1 A*02:01
GILGFVFTL Neg Ctrl 2 A*02:01 FLLTRILTI Neg Ctrl 3 G9 A*24:02
TYGPVFMCL Neg Ctrl 1 A*24:02 RYLKDQQLL Neg Ctrl 2 A*24:02 PYLFWLAAI
Neg Ctrl 3 G10 A*01:01 ASSLPTTMNY MAGE3/6 Target A*01:01
YSEHPTFTSQY Neg Ctrl 1 A*01:01 VSDGGPNLY Neg Ctrl 2 A*01:01
ATDALMTGY Neg Ctrl 3 G11 (=G3) A*11:01 IVTDFSVIK Neg Ctrl 1 A*11:01
KSMREEYRK Neg Ctrl 2 A*11:01 SSCSSCPLSK Neg Ctrl 3 G12 (=G6)
A*03:01 RLRAEAQVK Neg Ctrl 1 A*03:01 RLRPGGKKK Neg Ctrl 2 A*03:01
QVPLRPMTYK Neg Ctrl 3
Generation and Stability Analysis of HLA-PEPTIDE Target Complexes
and Counterscreen Peptide-HLA Complexes
[0774] Results for the G5 counterscreen "minipool" and G2 target
are shown in FIG. 5. All three counterscreen peptides and the G5
peptide rescued the HLA complex from dissociation.
[0775] Results for the additional G5 "complete" pool counterscreen
peptides are shown in FIG. 6, demonstrating that they also form
stable HLA-peptide complexes.
[0776] Results for counterscreen peptides and G8 target are shown
in FIG. 7. All three counterscreen peptides and the G8 peptide
rescued the HLA complex from dissociation.
[0777] Results for the G10 counterscreen "minipool" and G10 target
are shown in FIG. 8. All three counterscreen peptides and the G10
peptide rescued the HLA complex from dissociation.
[0778] Results for the additional G8 and G10 "complete" pool
counterscreen peptides are shown in FIG. 9, demonstrating that they
also form stable HLA-peptide complexes.
[0779] Phage Library Screening
[0780] The highly diverse SuperHuman 2.0 synthetic naive scFv
library from Distributed Bio Inc was used as input material for
phage display, which has a 7.6.times.10.sup.10 total diversity on
ultra-stable and diverse VH/VL scaffolds. For both screen 1 (see
FIG. 3) and screen 2 (see FIG. 4) three to four rounds of
bead-based phage panning with the target pHLA complex (as shown in
Table 3) were conducted using established protocols to identify
scFv binders to pHLAs G5, G8 and G10, respectively. For each round
of panning, the phage library was initially depleted with 18 pooled
negative pHLA complexes prior to the binding step with the target
pHLAs. The phage titer was determined at every round of panning to
establish removal of non-binding phage. The output phage
supernatant was also tested for target binding by ELISA and
suggested progressive enrichment of G5-, G8 and G10 binding phage
(see FIG. 10).
TABLE-US-00035 TABLE 3 Phage library screening strategy Antigen
Round concentration Washes R1 100 pmol 3X PBST + 3X PBS (5 min
washes) R2 25 pmol 5 PBST (2x 30 sec, 3x 5 min) + 5 PBS (2x 30 sec,
3x 5 min) R3 10 pmol 8 PBST (4x 30 sec, 4x 5 min) + 8 PBS (4x 30
sec, 4x 5 min) R4 5 pmol, 10 pmol 30 min PBST + 30 min PBS
[0781] Bacterial periplasmic extracts (PPEs) of individual output
clones were subsequently generated in 96-well plates using
well-established protocols. The PPEs were used to test for binding
to the target pHLA antigen by high throughput PPE ELISA. Positive
clones were sequenced and re-arrayed to select sequence-unique
clones. Sequence unique clones were then tested in a secondary
ELISA for binding to target pHLA versus the panel of HLA-matched
negative control pHLA complexes, thus establishing target
specificity. The G8 negative control HLA complexes (i.e. A*24:02)
did not HLA-match with the G8 target HLA complex (i.e. A*02:01).
Therefore, HLA-A*02:01 complexes presenting the peptides LLFGYPVYV,
GILGFVFTL or FLLTRILTI from G7 were used as HLA-matched minipool of
negative controls for G8 in further biochemical and functional
characterization assays for the TCR-mimetic Abs retrieved from the
scFv library.
[0782] Isolation of scFv Hits
[0783] Individual, soluble scFv protein fragments were produced and
purified for the scFv clones that were found to be selective when
expressed in PPEs. As shown by scFv PPE ELISA, these clones
exhibited at least three-fold selective binding to the target pHLA
as compared to binding to the minipool of negative control pHLAs.
Soluble scFv production allowed for further biochemical and
functional characterization.
[0784] The resulting VH and VL sequences for the scFvs that bind
target G5 are shown in Table 4. To clarify the organization of
Table 4, and other Tables of scFv sequences, each scFv was assigned
a clone name. For all clone names, clone names recite the target
(e.g., G5), the plate number (e.g., plate 7), and well number
(e.g., well E7) of the 96-well plate from which the clone was
originally picked. For example, clone names G5-P7E07, G5-7E7,
G5(7E7), G5(7E07) G5_P7_E7, all refer to the same scFv clone. For
example, Table 4 indicates that the scFv from clone G5_P7_E7 has
the VH sequence
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGIINPRSG
STKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGVRYYGMDVWG QGTTVTVSSAS
and the VL sequence
TABLE-US-00036 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTP
ITFGQGTRLEIK.
[0785] The resulting CDR sequences for the scFvs that bind target
G5 are shown in Table 5. To clarify the organization of Table 5,
each scFv was assigned a clone name in Table 5. For example, the
scFv from clone G5_P7_E7 has an HCDR1 sequence that is YTFTSYDIN,
an HCDR2 sequence that is GIINPRSGSTKYA, an HCDR3 sequence that is
CARDGVRYYGMDVW, an LCDR1 sequence that is RSSQSLLHSNGYNYLD, an
LCDR2 sequence that is LGSYRAS, and an LCDR3 sequence that is
CMQGLQTPITF, according to the Kabat numbering system.
[0786] The resulting VH and VL sequences for the scFvs that bind
target G8 are shown in Table 6. Table 6 is organized similarly to
Table 4.
[0787] The resulting CDR sequences for the scFvs that bind target
G8 are shown in Table 7. Table 7 is organized similarly to Table
5.
[0788] The resulting VH and VL sequences for the scFvs that bind
target G10 are shown in Table 8. Table 8 is organized similarly to
Table 4.
[0789] The resulting CDR sequences for the scFvs that bind target
G8 are shown in Table 9. Table 9 is organized similarly to Table
5.
[0790] A number of clones were formatted into scFv, Fab, and IgG to
facilitate biochemical, structural, and functional characterization
(see Table 10).
TABLE-US-00037 TABLE 10 Hit rate of the screening campaigns. Clones
were reformatted into (a) IgG for biochemical and functional
characterization, (b) Fab constructs for protein crystallography
and HDX mass spectrometry, and (c) scFv constructs for HDX mass
spectrometry. Group G5 G8 G10 HLA B*35:01 A*02:01 A*01:01 Peptide
MAGEA6 FOXE1 MAGE3/6 Sequence Unique 81 17 23 Binders Selective
Binders 18 17 18 IgG 18 17 18 Fab 4 3 2 scFv 8 7 6
[0791] FIG. 11 depicts a flow chart describing the antibody
selection process, including criteria and intended application for
the scFv, Fab, and IgG formats. Briefly, clones were selected for
further characterization based on sequence diversity, binding
affinity, selectivity, and CDR3 diversity.
[0792] To assess sequence diversity, dendrograms were produced
using clustal software. The predicted 3D structures of the scFv
sequences, based on the VH type, were also taken into
consideration. Binding affinity as determined by the equilibrium
dissociation constant (K.sub.D) was measured using an Octet HTX
(ForteBio). Selectivity for the specific peptide-HLA complexes was
determined with an ELISA titration of the purified scFvs as
compared to the minipool of negative control pHLA complexes or
streptavidin alone. Cutoff values for the K.sub.D and selectivity
were determined for each target set based on the range of values
obtained for the Fabs within each set. Final clones were selected
based on diversity in sequence families and CDR3 sequences.
[0793] The overall number of hits following phage library screening
and scFv isolation are listed in Table 10, above.
[0794] Materials and Methods
[0795] HLA Expression and Purification:
[0796] Recombinant proteins were obtained through bacterial
expression using established procedures (Garboczi, Hung, &
Wiley, 1992). Briefly, the .alpha. chain and .beta.2 microglobulin
chain of various human leukocyte antigens (HLA) were expressed
separately in BL21 competent E. Coli cells (New England Biolabs).
Following auto-induction, cells were lysed via sonication in
Bugbuster.RTM. plus benzonase protein extraction reagent (Novagen).
The resulting inclusion bodies were washed and sonicated in wash
buffer with and without 0.5% Triton X-100 (50 mM Tris, 100 mM NaCl,
1 mM EDTA). After the final centrifugation, inclusion pellets were
dissolved in urea solution (8 M urea, 25 mM MES, 10 mM EDTA, 0.1 mM
DTT, pH 6.0). Bradford assay (Biorad) was used to quantify the
concentration and the inclusion bodies were stored at -80.degree.
C.
[0797] Refold of pHLA and Purification:
[0798] HLA complexes were obtained by refolding of recombinantly
produced subunits and a synthetically obtained peptide using
established procedures. (Garboczi et al., 1992) Briefly, the
purified .alpha. and .beta.2 microglobulin chains were refolded in
refold buffer (100 mM Tris pH 8.0, 400 mM L-Arginine HCl, 2 mM
EDTA, 50 mM oxidized glutathione, 5 mM reduced glutathione,
protease inhibitor tablet) with either the target peptide or a
cleavable ligand. The refold solution was concentrated with a
Vivaflow 50 or 50R crossflow cassette (Sartorius Stedim). Three
rounds of dialyses in 20 mM Tris pH 8.0 were performed for at least
8 hours each. For the antibody screening and functional assays, the
refolded HLA was enzymatically biotinylated using BirA biotin
ligase (Avidity). Refolded protein complexes were purified using a
HiPrep (16/60 Sephacryl 5200) size exclusion column attached to an
AKTA FPLC system. Biotinylation was confirmed in a streptavidin
gel-shift assay under non-reducing conditions by incubating the
refolded protein with an excess of streptavidin at room temperature
for 15 minutes prior to SDS-PAGE. The peptide-HLA complexes were
aliquoted and stored at -80.degree. C.
[0799] Peptide Exchange:
[0800] HLA-peptide stability was assessed by conditional ligand
peptide exchange and stability ELISA assay. Briefly, conditional
ligand-HLA complexes were subjected to conditional stimulus in the
presence or absence of the counterscreen or test peptides. Exposure
to the conditional stimulus cleaves the conditional ligand from the
HLA complex, resulting in dissociation of the HLA complex. If the
counterscreen or test peptide stably binds the .alpha.1/.alpha.2
groove of the HLA complex, it "rescues" the HLA complex from
disassociation. In short, a mixture of 100 .mu.L of 50 .mu.M of the
novel peptide (Genscript) and 0.5 .mu.M recombinantly produced
cleavable ligand-loaded HLA in 20 mM Tris HCl and 50 mM NaCl at pH
8 was placed on ice. The mixture was irradiated for 15 min in a UV
cross-linker (CL-1000, UVP) equipped with 365-nm UV lamps at
.about.10 cm distance.
[0801] MHC Stability Assay:
[0802] The MHC stability ELISA was performed using established
procedures. (Chew et al., 2011; Rodenko et al., 2006) A 384-well
clear flat bottom polystyrene microplate (Corning) was precoated
with 50 .mu.l of streptavidin (Invitrogen) at 2 .mu.g/mL in PBS.
Following 2 h of incubation at 37.degree. C., the wells were washed
with 0.05% Tween 20 in PBS (four times, 50 .mu.L) wash buffer,
treated with 50 .mu.l of blocking buffer (2% BSA in PBS), and
incubated for 30 min at room temperature. Subsequently, 25 .mu.l of
peptide-exchanged samples that were 300.times. diluted with 20 mM
Tris HCl/50 mM NaCl were added in quadruplicate. The samples were
incubated for 15 min at RT, washed with 0.05% Tween wash buffer
(4.times.50 L), treated for 15 min with 25 .mu.L of HRP-conjugated
anti-02m (1 .mu.g/mL in PBS) at RT, washed with 0.05% Tween wash
buffer (4.times.50 .mu.L), and developed for 10-15 min with 25 L of
ABTS-solution (Invitrogen). The reactions were stopped by the
addition of 12.5 .mu.L of stop buffer (0.01% sodium azide in 0.1 M
citric acid). Absorbance was subsequently measured at 415 nm using
a spectrophotometer (SpectraMax i3x; Molecular Devices).
[0803] Phage Panning:
[0804] For each round of panning, an aliquot of starting phage was
set aside for input titering and the remaining phage was depleted
three times against Dynabead M-280 streptavidin beads (Life
Technologies) followed by a depletion against Streptavidin beads
pre-bound with 100 pmoles of pooled negative peptide-HLA complexes.
For the first round of panning, 100 pmoles of peptide-HLA complex
bound to streptavidin beads was incubated with depleted phage for 2
hours at room temperature with rotation. Three five-minute washes
with 0.5% BSA in 1.times.PBST (PBS+0.05% Tween-20) followed by
three five-minute washes with 0.5% BSA in 1.times.PBS were utilized
to remove any unbound phage to the peptide-HLA complex bound beads.
To elute the bound phage from the washed beads, 1 mL 0.1M TEA was
added and incubated for 10 minutes at room temperature with
rotation. The eluted phage was collected from the beads and
neutralized with 0.5 mL 1M Tris-HCl pH 7.5. The neutralized phage
was then used to infect log growth TG-1 cells (OD.sub.600=0.5) and
after an hour of infection at 37.degree. C., cells were plated onto
2YT media with 100 .mu.g/mL carbenicillin and 2% glucose (2YTCG)
agar plates for output titer and bacterial growth for subsequent
panning rounds. For subsequent rounds of panning, selection antigen
concentrations were lowered while washes increased by amount and
length of wash times at show in Table 3.
[0805] Input Output Phage Titer:
[0806] Each round of input titer was serially diluted in 2YT media
to 10.sup.10. Log phase TG-1 cells are infected with diluted phage
titers (10.sup.7-10.sup.10) and incubated at 37.degree. C. for 30
minutes without shaking followed by another 30 minutes with gentle
shaking. Infected cells are plated onto 2YTCG plates and incubated
overnight at 30.degree. C. Individual colonies were counted to
determine input titer. Output titers were performed following 1 h
infection of eluted phage into TG-1 cells. 1, 0.1, 0.01, and 0.001
.mu.L of infected cells were plated onto 2YTCG platers and
incubated overnight at 30.degree. C. Individual colonies were
counted to determine output titer.
[0807] Selective Target Binding of Bacterial Periplasmic
Extracts:
[0808] For scFv PPE ELISAs, 96-well and/or 384-well streptavidin
coated plates (Pierce) were coated with 2 .mu.g/mL peptide-HLA
complex in HLA buffer and incubated overnight at 4.degree. C.
Plates were washed three times between each step with PBST
(PBS+0.05% Tween-20). The antigen coated plates were blocked with
3% BSA in PBS (blocking buffer) for 1 hour at room temperature.
After washing, scFv PPEs were added to the plates and incubated at
room temperature for 1 hour. Following washing, mouse anti-v5
antibody (Invitrogen) in blocking buffer was added to detect scFv
and incubated at room temperature for 1 hour. After washing,
HRP-goat anti-mouse antibody (Jackson ImmunoResearch) was added and
incubated at room temperature for 1 hour. The plates were then
washed three times with PBST and 3 times with PBS before HRP
activity was detected with TMB 1-component Microwell Peroxidase
Substrate (Seracare) and neutralized with 2N sulfuric acid.
[0809] For negative peptide-HLA complex counterscreening, the scFv
PPE ELISAs were performed as described above, except for the
coating antigen. Namely, the HLA mini-pools (see Tables 1 and 2)
were used that consisted of 2 .mu.g/mL of each of the three
negative peptide-HLA complexes pooled and coated onto streptavidin
plates for comparison binding to their particular pHLA complex.
Alternatively, HLA complete pools consisted of 2 .mu.g/mL of each
of all 18 negative peptide-HLA complexes pooled together and coated
onto streptavidin plates for comparison binding to their particular
pHLA complex.
[0810] Construction and Production of scFv Protein Fragments:
[0811] The expression plasmid was transformed into BL21(DE3) strain
and co-expressed with a periplasmic chaperone in a 400 mL E. coli
culture. The cell pellet was reconstituted as follows: 10 mL/1 g
biomass with (25 mM HEPES, pH7.4, 0.3M NaCl, 10 mM MgCl2, 10%
glycerol, 0.75% CHAPS, 1 mM DTT) plus lysozyme, and benzonase and
Lake Pharma protease inhibitor cocktail. The cell suspension was
incubated on a shaking platform at RT for 30 minutes. Lysates were
clarified by centrifugation at 4.degree. C., 13,000.times.rpm for
15 min. The clarified lysate was loaded onto 5 mL of Ni NTA resin
pre-equilibrated in IMAC Buffer A (20 mM Tris-HCl, Ph7.5; 300 mM
NaCl/10% Glycerol/1 mM DTT). The resin was washed with 10 column
volumes (CVs) of Buffer A (or until a stable baseline was reached),
followed by 10 CVs of 8% IMAC Buffer B (20 mM Tris-HCl, Ph7.5; 300
mM NaCl/10% Glycerol/1 mM DTT/250 mM Imidazole). The target protein
was eluted in a 20CV gradient to 100% IMAC Buffer B. The column was
washed with 5CVs of 100% IMAC B to ensure complete protein removal.
Elution fractions were analyzed by SDS-PAGE and Western blot
(anti-His) and pooled accordingly. The pool was dialyzed with the
final formulation buffer (20 mM Tris-HCl, Ph7.5; 300 mM NaCl/10%
glycerol/1 mM DTT), concentrated to a final protein concentration
>0.3 mg/mL, aliquoted into 1 mL vials, and flash frozen in
liquid nitrogen. Final QC steps included SDS-PAGE and A280
absorbance measurements.
[0812] Construction and Production of Fab Protein Fragments:
[0813] The constructs of selected G5, G8 and G10 Fabs were cloned
into a vector optimized for mammalian expression. Each DNA
construct was scaled up for transfection and sequences were
confirmed. A 100 mL transient production was completed in HEK293
cells (Tuna293.TM. Process) for each. The proteins were purified by
anti-CH1 purification subsequently purified by size exclusion
chromatography (SEC) via HiLoad 16/600 Superdex 200. The mobile
phase used for SEC-polishing was 20 mM Tris, 50 mM NaCl, pH 7.
Final confirmatory CE-SDS analysis was performed.
[0814] Construction and Production of IgG Proteins:
[0815] The expression constructs of the G series antibodies were
cloned into a vector optimized for mammalian expression. Each DNA
construct was scaled up for transfection and sequences were
confirmed. A 10 mL transient production was completed in HEK293
cells (Tuna293.TM. Process) for each. The proteins were purified by
Protein A purification and final CE-SDS analysis was performed.
Example 4: Affinity of Fab Clones for their Respective HLA-PEPTIDE
Targets
[0816] Fab-formatted antibodies allow for accurate assessment of
monomeric binding to their respective HLA-PEPTIDE targets, while
avoiding confounding effects of bivalent interactions with the IgG
antibody format. Binding affinity was assessed by bio-layer
interferometry (BLI) using an Octet Qke (ForteBio). Briefly,
biotinylated pHLA complexes in kinetics buffer were loaded onto
streptavidin sensors for 300 seconds, at concentrations which gave
the optimal nm shift response (approximately 0.6 nm) for each Fab
at the highest concentration used. The ligand-loaded tips were
subsequently equilibrated in the kinetics buffer for 120 seconds.
The ligand-loaded biosensors were then dipped for 200 seconds in
the Fab solution titrated into 2-fold dilutions. Starting Fab
concentrations ranged from 100 nM to 2 .mu.M, iteratively optimized
based on the K.sub.D values of the Fab. The dissociation step in
the kinetics buffer was measured for 200 seconds. Data were
analyzed using the ForteBio data analysis software using a 1:1
binding model.
[0817] Results for HLA-PEPTIDE targets HLA-1B*35:01_EVDPIGHVY,
HLA-A*02:01 AIFPGAVPAA, and HLA-A*01:01 ASSLPTTMNY are shown in
Table 11, below.
TABLE-US-00038 TABLE 11 Optimized Octet BLI affinity measurements
of Fabs binding to their target peptide-HLA complex Kon Kdis Full
Target Fab clone KD (M) (1/Ms) (1/s) RA{circumflex over ( )}2 G5
G5-P7A05 1.19E-07 4.10E+05 4.87E-02 0.997 G5 G5-P7B3 2.54E-07
4.42E+05 9.09E-02 0.993 G5 G5-P7E7 2.82E-08 9.02E+05 2.48E-02 0.991
G5 G5-P7F6 3.37E-08 9.15E+05 3.06E-02 0.995 G5 G5-P1C12 8.59E-08
4.93E+03 1.58E-02 0.983 G8 G8-P4F05 9.84E-07 6.40E+04 6.30E-02
0.976 G8 G8-P1B03 3.07E-07 1.67E+05 5.11E-02 0.996 G8 G8-P5G08
5.30E-07 9.97E+04 5.28E-02 0.927 G8 R3G8-P2C10 1.77E-08 7.50E+04
1.30E-03 0.997 G8 R3G8-P1C11 1.78E-07 1.90E+05 3.38E-02 0.997 G8
R3G8-P2E04 2.86E-07 5.45E+05 7.89E-02 0.842 G10 R3G10-P1B07
3.75E-08 1.65E+05 6.15E-03 0.997 G10 R3G10-P4E07 4.28E-07 4.77E+05
1.11E-01 0.990
[0818] FIGS. 12A, 121B, and 12C depicts BLI results for Fab clone
G5-P7A05 to HLA-PEPTIDE target B*35:01-EVDPGHVY (12A), Fab clones
R3G8-P2C10 and G8-P1C11 to HLA-PEPTIDE target A*02:01-AFPGAVPAA
(12B, P2C10 on left and P3C11 on right), and Fab clone R3G10-P1B07
to HLA-PEPTIDE target A*01:01-ASSLPTTMNY (12C), respectively.
[0819] FIGS. 71A and 71B show LI results for G2 target Fab clone
G2-P1H11 and for G7 target Fab clone G7R4-B5-P2E9, respectively.
FIG. 90 shows L results for G2 target Fab clone G2-P2C06.
[0820] Results are shown in the Table below.
TABLE-US-00039 TABLE 43 Optimized Octet BLI affinity measurements
of Fabs binding to their target peptide-HLA complex Kon Full Target
Fab clone KD (M) (1/Ms) Kdis (1/s) R{circumflex over ( )}2 G2
G2-P1B06 4.44E-08 1.06E+06 3.23E-02 0.991 G2 G2-P2A03 1.09E-07
3.32E+05 3.60E-02 0.998 G2 G2-P1B12 2.28E-08 3.66E+05 7.28E-03
0.980 G2 G2-P2A11 2.81E-08 6.33E+05 1.72E-02 0.992 G2 G2-P1H01
1.55E-08 9.52E+05 1.48E-02 0.984 G2 G2-P1H11 4.99E-08 5.81E+05
2.80E-02 0.994 G2 G2-P2C06 3.06 E-08 1.14 E+06 3.48 E-02 0.992 G7
2-G7R4-P2C2 5.31E-07 1.04E+05 5.43E-02 0.986 G7 3-G7R4-P1A3
5.32E-07 1.97E+05 9.94E-02 0.988 G7 4-G7R4-B5- 1.18E-08 1.85E+05
2.12E-03 0.992 P2E9
[0821] FIG. 105 shows BLI results for G8 target Fab clones
G8-P4F05, G8-P1B03, and G8-P5G08 to HLA-PEPTIDE target
A*02:01-AIFPGAVPAA; as well as BLI results for G5 target Fab clone
G5-P1C12 to HLA-PEPTIDE target B*35:01-EVDPIGHVY.
[0822] The Fab-formatted antibodies bind to their respective
HLA-PEPTIDE targets with high affinity.
Example 5: Positional Scanning of G2, G5, G7, G8, and G10
Restricted Peptide Sequences
[0823] Positional scanning of the G2, G5, G7, G8, and G10
restricted peptides was carried out to determine the amino acid
residues which act as contact points for selected Fab clones or
residues that impact, directly or indirectly, the interaction of
the HLA-PEPTIDE target with the Fab.
[0824] FIG. 13 depicts a first experimental design for the
positional scanning experiments. Positional scanning libraries of
variant G2, G5, G7, G8, and G10 restricted peptides were generated
with amino acid substitutions at a single position in the
restricted peptide sequence, scanning across all positions. The
amino acid substitutions at a given position were either alanine
(conservative substitution), arginine (positively charged), or
aspartate (negatively charged).
[0825] Peptide-HLA complexes comprising the positional scanning
library members and the HLA subtype allele were generated as
described in Example 3. Stability of the resulting complexes was
determined using conditional ligand peptide exchange and stability
ELISA as described in Example 3. Such stability analysis may
identify residues on the restricted peptide which are important for
binding and stabilizing the HLA molecule. Binding affinity of the
selected Fab clone to the variant peptide-HLA complexes was
assessed by BLI as described in Example 4. Positional variants that
result in stable HLA complex formation and weakened Fab binding may
identify residues that are likely involved, directly or indirectly,
in determining the interaction of the peptide-HLA complex with the
Fab clone. For instance, the identified residues may form part of
the epitope that binds the ABP, or alternatively may influence the
conformational shape or presentation of the epitope.
[0826] FIG. 14A depicts stability results for the G5 positional
variant-HLAs, indicating that the majority of peptide mutations
does not impact binding of those peptides to the relevant pHLA.
[0827] FIG. 14B depicts binding affinity of Fab clone G5-P7A05 to
the G5 positional variant-HLAs, indicating positions P2-P8 of the
restricted peptide as likely involved, directly or indirectly, in
determining the interaction of the peptide-HLA complex with the Fab
clone.
[0828] FIG. 15A depicts stability results for the G8 positional
variant-HLAs, indicating that positions P2, P7 and P10 were not
amenable to substitution with the Arg- or Asp-residue and therefore
are likely to be important for the peptide to bind the HLA
protein.
[0829] FIG. 15B depicts binding affinity of Fab clone G8-P2C10 to
the G8 positional variant-HLAs, indicating positions P1-P5 of the
restricted peptide as likely involved, directly or indirectly, in
determining the interaction of the peptide-HLA complex with the Fab
clone.
[0830] FIG. 46 depicts binding affinity of Fab clone G8-P1C11 to
the G8 positional variant-HLAs, indicating positions P3-P6 of the
restricted peptide as likely involved, directly or indirectly, in
determining the interaction of the peptide-HLA complex with the Fab
clone.
[0831] FIG. 16A depicts stability results for the G10 positional
variant-HLAs, indicating that positions 2, 5, 8, and 10 were not
amenable to amino acid substitution and therefore are likely to be
important for the peptide to bind the HLA protein.
[0832] FIG. 16B depicts binding affinity of Fab clone G10-P1B07 to
the G10 positional variant-HLAs, indicating positions P4, P6, and
P7 of the restricted peptide as likely involved, directly or
indirectly, in determining the interaction of the peptide-HLA
complex with the Fab clone.
[0833] A map of the amino acid substitutions for the positional
scanning experiments for G2 and G7 restricted peptides is shown in
FIG. 72. Asterisks denote lack of amino acid substitution.
[0834] FIG. 73A depicts stability results for the G2 positional
variant-HLAs, indicating that positions 2, 3, and 9 were not
amenable to amino acid substitutions and therefore are likely to be
important for the peptide to bind the HLA protein.
[0835] FIG. 73B depicts binding affinity of Fab clone G2-P1H11 to
the G2 positional variant-HLAs, indicating positions 3-9 of the
restricted peptide as likely involved, directly or indirectly, in
determining the interaction of the peptide-HLA complex with the Fab
clone.
[0836] FIG. 91A depicts stability results from a second experiment
for the G2 positional variant-HLAs, further confirming that
positions 2, 3, and 9 were not amenable to amino acid substitutions
and therefore are likely to be important for the peptide to bind
the HLA protein.
[0837] FIG. 91B depicts binding affinity of Fab clone G2-P2C06 to
the G2 positional variant-HLAs, indicating positions 7-8 of the
restricted peptide as likely involved, directly or indirectly, in
determining the interaction of the peptide-HLA complex with the Fab
clone.
[0838] FIG. 74A depicts stability results for the G7 positional
variant-HLAs, indicating that positions 1, 2, 6, and 9 were not
amenable to amino acid substitutions and therefore are likely to be
important for the peptide to bind the HLA protein.
[0839] FIG. 74B depicts binding affinity of Fab clone G7R4-B5-P2E9
to the G7 positional variant-HLAs, indicating positions 1-5 of the
restricted peptide as likely involved, directly or indirectly, in
determining the interaction of the peptide-HLA complex with the Fab
clone.
[0840] In a second positional scanning experiment, libraries of
variant restricted peptides for each HLA-PEPTIDE target were
generated, wherein each variant differed from the corresponding
HLA-PEPTIDE target by 1-3 amino acids. Binding affinity of the
selected Fab clone to the variant peptide-HLA complexes was
assessed by BLI as described above. Variants that weakened Fab
binding may identify residues that are likely involved, directly or
indirectly, in determining the interaction of the peptide-HLA
complex with the Fab clone
[0841] Data from the second positional scanning experiment,
assessing binding capability of G2P1H11 ABP to the library of
A*01:01_NTDNNLAVY variants, revealed that several single or
multiple variants at positions 4-9 were able to eliminate
detectable binding by biolayer interferometry (BLI) (data not
shown). These data were largely consistent with the first
positional scanning experiment described above.
[0842] Data from the second positional scanning experiment,
assessing binding capability of G10P3E04 ABP to the library of
A*01:01_ASSLPTTMNY variants, revealed that variant peptides which
failed to bind G10P3E04 comprised 2 amino acid differences compared
to the target peptide across positions 6-9. These data support the
notion that peptide positions 6-9 are important directly or
conformationally for binding to this target.
Example 6: Antibodies Bind Cells Presenting HLA-PEPTIDE Target
Antigens HLA-B*35:01 EVDPIGHVY, HLA-A*02:01 AIFPGAVPAA, and
HLA-A*01:01 ASSLPTTMNY
[0843] To verify that the identified TCR-like antibodies bind their
pHLA target G5, G8 and G10 in their natural context, e.g., on the
surface of antigen-presenting cells, selected clones were
reformatted to IgG and used in binding experiments with K562 cells
expressing the cognate HLA-PEPTIDE target. Briefly, cells were
transduced with either HLA-B*35:01 for the G5 target peptide,
HLA-A*02:01 for the G7 and G8 target peptides, or HLA-A*01:01 for
the G2 and G10 target peptides. The cells were then exogenously
pulsed with target or negative control peptide, e.g., as specified
in Tables 1 and 2, using established methods to generate the
relevant pHLA complexes on the cell surface.
[0844] Materials and Methods
[0845] Retroviral Production
[0846] The Phoenix-AMPHO cells (ATCC.RTM., CRL-3213.TM.) were
cultured in DMEM (Corning.TM., 17-205-CV) supplemented with 10% FBS
(Seradigm, 97068-091) and Glutamax (Gibco.TM., 35050079). K-562
cells (ATCC.RTM., CRL-243.TM.) were cultured in IMDM (Gibco.TM.,
31980097) supplemented with 10% FBS. Lipofectamine LTX PLUS (Fisher
Scientific, 15338100) contains a Lipofectamine reagent and a PLUS
reagent. Opti-MEM (Gibco.TM., 31985062) was purchased from Fisher
Scientific.
[0847] Phoenix cells were plated at 5.times.10.sup.5 cells/well in
a 6 well plate and incubated overnight at 37.degree. C. For the
transfection, 10 .mu.g plasmid, 10 .mu.L Plus reagent and 100 .mu.L
Opti-MEM were incubated at room temperature for 15 minutes.
Simultaneously, 8 .mu.L Lipofectamine was incubated with 92 .mu.L
Opti-MEM at room temperature for 15 minutes. These two reactions
were combined and incubated again for 15 minutes at room
temperature after which 800 .mu.L Opti-MEM was added. The culture
media was aspirated from the Phoenix cells and they were washed
with 5 mL pre-warmed Opti-MEM. The Opti-MEM was aspirated from the
cells and the lipofectamine mixture was added. The cells were
incubated for 3 hours at 37.degree. C. and 3 mL complete culture
medium was added. The plate was then incubated overnight at
37.degree. C. The media was replaced with Phoenix culture medium
and the plate incubated an additional 2 days at 37.degree. C.
[0848] The media was collected and filtered through a 45 .mu.m
filter into a clean 6 well dish. 20 .mu.L Plus reagent was added to
each virus suspension and incubated at room temperature for 15
minutes followed by the addition of 8 .mu.L/well of Lipofectamine
and another 15 min room temperature incubation.
[0849] K562 Cell Line Generation (Retroviral Transduction with
HLA)
[0850] K562 cells were counted and resuspended to 5E6 cells/mL and
100 .mu.L added to each virus suspension. The 6 well plate was
centrifuged at 700 g for 30 minutes and then incubated at
37.degree. C. for 5-6 hours. The cells and virus suspension were
then transferred to a T25 flask and 7 mL K562 culture medium was
added. The cells were then incubated for three days. The transduced
K562 cells were then cultured in medium supplemented with 0.6
.mu.g/mL Puromycin (Invivogen, ant-pr-1) and selection monitored by
flow cytometry.
[0851] Flow Cytometry Methods:
[0852] HLA-transduced K562 cells were pulsed the night before with
50 .mu.M of peptide (Genscript) in IDMEM containing 1% FBS in 6
well plates and incubated under standard tissue culture conditions.
Cells were harvested, washed in PBS, and stained with eBioscience
Fixable Viability Dye eFluor 450 for 15 minutes at room
temperature. Following another wash in PBS+1.sup.-2% FBS, cells
were resuspended with IgGs at varying concentrations. Cells were
incubated with antibodies for 1 hour at 4.degree. C. After another
wash, PE-conjugated goat anti-human IgG secondary antibody (Jackson
ImmunoResearch) was added at 1:100 to 1:200 for 30 minutes at
4.degree. C. After washing in PBS+1-2% FBS, cells were resuspended
in PBS+1-2% FBS and analyzed by flow cytometry. Flow cytometric
analysis was performed on the Attune NxT Flow Cytometer
(ThermoFisher) using the Attune NxT Software. Data were analyzed
using FlowJo.
[0853] Results
[0854] Four representative examples of antibody binding to either
G5-, G8- or G10-presenting K562 cells, as detected by flow
cytometry, are shown in FIGS. 17A, 17B, and 17C. Antibody binding
was observed in a dose-dependent manner that was selective for the
relevant target peptides.
[0855] In another flow cytometry experiment, HLA-transduced K562
cells were pulsed with 50 .mu.M of target or control peptides as
listed in Table 1 for G5 and in Table 2 for G8 and G10, and
pHLA-specific antibodies were detected by flow cytometry.
HLA-transduced K562 cells were pulsed with 50 .mu.M of target or
negative control peptides and antibody binding histograms were
plotted for G5-P7A05 at 20 .mu.g/mL, G8-2C10 at 30 .mu.g/mL,
G10-P1B07 at 30 .mu.g/mL, and G8-P1C11 at 30 .mu.g/mL. Histograms
are depicted in FIG. 18 and FIG. 47.
[0856] Results are shown in FIGS. 75 and 76. Both G2-P1H11 and
G7R4-B5-P2E9 selectively bound HLA-transduced K562 cells pulsed
with the target peptide, as compared to HLA-transduced cells pulsed
with the negative control peptides.
[0857] In another flow cytometry experiment, HLA-B*35:01-transduced
K562 cells were pulsed with 50 .mu.M of target peptide EVDPIGHVY
("EVD") or negative control peptide IPSINVHHY ("IPS"), and
pHLA-specific antibodies were detected by flow cytometry. Results
for G5 antibodies G5-7A05 and G4-1C12 are shown in FIG. 102.
Antibody binding was observed at all doses in a manner that was
selective for the target peptide.
[0858] In another flow cytometry experiment, HLA-A*02:01-transduced
K562 cells were pulsed with 50 .mu.M of target peptide AIFPGAVPAA
("AIF") or negative control peptide FLLTRILTI ("FLL"), and
pHLA-specific antibodies were detected by flow cytometry. Results
for G8 specific antibodies G8-1B03, G8-5G08, G8-4F05, G81C11,
G82C10, and G82C11 are shown in FIG. 103. Antibody binding was
observed at all doses in a manner that was selective for the target
peptide.
[0859] In another flow cytometry experiment, HLA-A*01:01-transduced
K562 cells were pulsed with 50 .mu.M of target peptide ASSLPTTMNY
("ASSL") or negative control peptide ATDALMTGY ("ATDA"), and
pHLA-specific antibodies were detected by flow cytometry. Results
for G10 specific antibodies G10-3E09 and G10-1H01 are shown in FIG.
104. Antibody binding was observed in a dose dependent manner in a
manner that was selective for the target peptide.
Example 7: Antibodies Bind to Tumor Cell Lines that Express the
Target Gene and HLA Subtype
[0860] Tumor cell lines were chosen based on expression of the HLA
subtype and target gene of interest, as assessed by a publicly
available database (TRON http://celllines.tron-mainz.de). The
selection of the tumor cell line for cell binding assays is shown
in Table 12 below.
TABLE-US-00040 TABLE 12 selection of tumor cell lines for cell
binding assay Cell line Target expression HLA type LN229 MAGEA6
(137.6 RPKM) B*35:01; B*35:01 (26.53 RPKM) (G5) BV173 FOXE1 (18.1
RPKM) A*30:01; A*02:01 (142.25 RPKM) (G8) Colo829 MAGEA3 (119.3
RPKM) A*01:01; A*0101 (143.7 RPKM) (G10) MAGEA6 (215.4 RPKM)
[0861] The LN229, BV173, and Colo829 tumor cell lines were
propagated under standard tissue culture conditions. Flow cytometry
was performed as described in Example 6. Cells were incubated with
30 .mu.g/mL or 0 .mu.g/mL antibody followed by PE conjugated
anti-human secondary IgG.
[0862] Results are depicted in FIG. 19. Panel A shows a histogram
plot for G5-P7A05 binding to glioblastoma line LN229. Panel B shows
a histogram plot for G8-P2C10 binding to leukemia line BV173. Panel
C shows a histogram plot for G10-P1B07 binding to CRC line
Colo829.
Example 8: Identification of TCRs that Bind HLA-PEPTIDE Target
HLA-A*01:01 ASSLPTTMNY or HLA-PEPTIDE Target HLA-A*01:01
HSEVGLPVY
[0863] Peripheral blood mononuclear cells (PBMCs) were obtained by
processing leukapheresis samples from healthy donors. Frozen PBMCs
were thawed and incubated with cocktail of biotinylated CD45RO,
CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD57, CD123, anti-HLA-DR,
CD235a (Glycophorin A), CD244, and CD4 antibodies and were
subsequently magnetically labeled with anti-biotin microbeads for
removal from PBMC population. Enriched naive CD8 T cells were
labelled with tetramers comprising target peptide and appropriate
MHC molecule, stained with live/dead and lineage markers and sorted
by flow cytometry cell sorter. Following polyclonal expansion, one
of two paths may be taken. If a large fraction of population is
specific for the HLA-PEPTIDE target, the T cell population may be
sequenced as a whole. Alternatively, the cells harboring TCRs
specific for the HLA-PEPTIDE target may be resorted, and only cells
isolated after resort are sequenced using 10.times. Genomics single
cell resolution paired immune TCR profiling approach.
[0864] Here, cells harboring TCRs specific for the HLA-PEPTIDE
target HLA-A*01:01 ASSLPTTMNY were resorted and sequenced as
described above. Specifically, two-to-eight thousand live T cells
were partitioned into single cell emulsions for subsequent single
cell cDNA generation and full-length TCR profiling (5' UTR through
constant region--ensuring alpha and beta pairing). This approach
utilized a molecularly barcoded template switching oligo at the 5'
end of the transcript. An alternative approach utilizes a
molecularly barcoded constant region oligo at the 3' end. Another
alternative approach couples an RNA polymerase promoter to either
the 5' or 3' end of a TCR. All of these approaches enable the
identification and deconvolution of alpha and beta TCR pairs at the
single-cell level. The resulting barcoded cDNA transcripts
underwent an optimized enzymatic and library construction workflow
to reduce bias and ensure accurate representation of clonotypes
within the pool of cells. Libraries were sequenced on Illumina's
MiSeq or HiSeq4000 instruments (paired-end 150 cycles) for a target
sequencing depth of about five to fifty thousand reads per
cell.
[0865] Sequencing reads were processed through the 10.times.
provided software Cell Ranger. Sequencing reads are tagged with a
Chromium cellular barcodes and UMIs, which are used to assemble the
V(D)J transcripts cell by cell. The assembled contigs for each cell
were then annotated by mapping the assembled contigs to the
Ensemble v87 V(D)J reference sequences. Clonotypes were defined as
alpha, beta chain pairs of unique CDR3 amino acid sequences.
Clonotypes were filtered for single alpha and single beta chain
pairs present at frequency above 2 cells to yield the final list of
clonotypes per target peptide in a specific donor.
[0866] Two different donors were analyzed over 6 experiments for
ASSLPTTMNY and 2 experiments for HSEVGLPVY targets. FIGS. 20A and
20B show the number of target-specific T cells isolated per
experiment and number of target-specific unique clonotypes
identified per experiment, respectively. Each color represent data
from one experiment.
[0867] Table 13 depicts the cumulative number of T cells and unique
TCRs identified across all experiments and average number of
target-specific T cells per 3 million of naive CD8 T cells.
TABLE-US-00041 TABLE 13 cumulative data from TCR identification
experiment Cumu- Average Cumu- lative fre- lative num- quency
number ber per 3E6 of of naive identified Target HLA isolated CD8 T
clono- sequence Gene restriction cells cells types ASSLPTTMNY MAGE
A*01:01 3516 464 550 A3/6 HSEVGLPVY DCAF12L1 A*01:01 1762 539
142
[0868] Annotated sequences of the identified TCR clonotypes
specific for HLA-PEPTIDE A*01:01_ASSLPTTMNY are shown in Table 14.
This table is included in PCT/US2018/06793, filed on Dec. 28, 2018,
which is incorporated by reference in its entirety.
[0869] Alpha and beta CDR3 sequences of the identified TCR
clonotypes specific for HLA-PEPTIDE A*01:01_ASSLPTTMNY are shown in
Table 15. For clarity, as in Table 14, each identified TCR was
assigned a TCR ID number. For example TCR ID #1 comprises the
.alpha.CDR3 sequence CAGPGNTGKLIF and the .beta.CDR3 sequence
CASSNAGDQPQHF.
[0870] Full length alpha V(J) and beta V(D)J sequences of the
identified TCR clonotypes specific for HLA-PEPTIDE
A*01:01_ASSLPTTMNY are shown in Table 16. For example TCR ID #1
comprises the alpha V(J) sequence
MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQ
RPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGPGN
TGKLIFGQGTTLQVK and the beta V(D)J sequence
TABLE-US-00042 MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDA
MYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSA
QKNPTAFYLCASSNAGDQPQHFGDGTRLSIL.
[0871] Annotated sequences of the identified TCR clonotypes
specific for HLA-PEPTIDE A*01:01_HSEVGLPVY are shown in Table 17.
This table is included in PCT/US2018/06793, filed on Dec. 28, 2018,
which is incorporated by reference in its entirety.
[0872] Alpha and beta CDR3 sequences of the identified TCR
clonotypes specific for HLA-PEPTIDE A*01:01 HSEVGLPVY are shown in
Table 18. This table is included in PCT/US2018/06793, filed on Dec.
28, 2018, which is incorporated by reference in its entirety.
[0873] Full length alpha V(J) and beta V(D)J sequences of the
identified TCR clonotypes specific for HLA-PEPTIDE A*01:01
HSEVGLPVY are shown in Table 19. This table is included in
PCT/US2018/06793, filed on Dec. 28, 2018, which is incorporated by
reference in its entirety.
Example 9: Identification of Antibodies or Antigen-Binding
Fragments Thereof that Bind HLA-PEPTIDE Complexes
[0874] Identification of Single-Chain Variable Fragment (scFv)
Antibodies Targeting MHC Class I Molecules Presenting Tumor
Antigens
[0875] Potent and selective single chain antibodies targeting human
class I MHC molecules presenting tumor antigens of interest are
identified using phage display. Phage libraries are prepared for
screening by removing non-specific class I MHC binders. Multiple
soluble human peptide-MHC (pMHC) molecules different from the
target pMHCs are utilized to pan pre-existing phage libraries to
remove scFvs that non-specifically bind class I MHC. To identify
scFvs that selectively bind pMHCs of interest, target pMHCs are
utilized for at least 1-3 rounds of panning with the prepared phage
library. scFv hits identified in the screen are then evaluated
against a panel of irrelevant pMHCs to identify scFv leads that
bind selectively to the target pMHCs. Lead scFvs are characterized
to determine target binding specificity and affinity. Lead scFvs
that demonstrate potent and selective binding are converted to
full-length IgG monoclonal antibody (mAb) constructs. In addition,
the lead scFvs are incorporated into bi-specific mAb constructs and
chimeric antigen receptor (CAR) constructs that can be used to
generate CAR T-cells. Full-length bi-specifics or scFV-based
bi-specifics can be constructed.
[0876] Demonstrate Targeting of Human Tumor Cells In Vitro
[0877] Immunohistochemistry techniques are utilized to demonstrate
specific binding of lead antibodies to human tumor cells or cell
lines expressing target pMHC molecules. T-cell lines transfected
with CAR-T constructs are incubated with human tumor cells to
demonstrate killing of tumor cells in vitro. Alternatively, tumor
cells expressing the target are incubated with bi-specific
constructs (encoding the ABP and an effector domain) and PBMCs or T
cells.
[0878] In Vivo Proof-of-Concept
[0879] Lead antibody or CAR-T constructs are evaluated in vivo to
demonstrate directed tumor killing in humanized mouse tumor models.
Lead antibody or CAR-T constructs are evaluated in xenograft tumor
models engrafted with human tumors and PBMCs. Anti-tumor activity
is measured and compared to control constructs to demonstrate
target-specific tumor killing.
[0880] Identification of Monoclonal Antibodies (mAbs) that Target
MHC Class I Molecules Presenting Tumor Antigens Using Rabbit B Cell
Cloning Technologies
[0881] Potent and selective mAbs targeting human class I MHC
molecules presenting tumor antigens of interest are identified.
Soluble human pMHC molecules presenting human tumor antigens are
utilized for multiple mouse or rabbit immunizations followed by
screening of B cells derived from the immunized animals to identify
B cells that express mAbs that bind to target class I MHC
molecules. Sequences encoding the mAbs identified from the mouse or
rabbit screens will be cloned from the isolated B cells. The
recovered mAbs are then evaluated against a panel of irrelevant
pMHCs to identify lead mAbs that bind selectively to the target
pMHCs. Lead mAbs will be fully characterized to determine target
binding affinity and selectivity. Lead mAbs that demonstrate potent
and selective binding are humanized to generate full-length human
IgG monoclonal antibody (mAb) constructs. In addition, the lead
mAbs are incorporated into bi-specific mAb constructs and chimeric
antigen receptor (CAR) constructs that can be used to generate CAR
T-cells. Full-length bi-specifics or scFV-based bi-specifics can be
constructed.
[0882] Demonstrate Targeting of Human Tumor Cells In Vitro
[0883] Immunohistochemistry techniques are utilized to demonstrate
specific binding of lead antibodies to human tumor cells expressing
target pMHC molecules. T-cell lines transfected with CAR-T
constructs are incubated with human tumor cells to demonstrate
killing of tumor cells in vitro. Alternatively, tumor cells
expressing the target are incubated with bi-specific constructs
(encoding the ABP and an effector domain) and PBMCs or T cells.
[0884] In Vivo Proof-of-Concept
[0885] Lead antibody or CAR-T constructs are evaluated in vivo to
demonstrate directed tumor killing in humanized mouse tumor models.
Lead antibody or CAR-T constructs are evaluated in xenograft tumor
models engrafted with human PBMCs. Anti-tumor activity is measured
and compared to control constructs to demonstrate target-dependent
tumor killing.
[0886] Potent and selective ABPs that selectively target human
class I MHC molecules presenting tumor antigens will be identified
using phage display or B cell cloning technologies. The utility of
the ABPs will be demonstrated by showing that the ABPs mediated
tumor cell killing in vitro and in vivo when incorporated into
antibody or CAR-T cell constructs.
Example 10: Identification of TCRs that Bind HLA-PEPTIDE
Complexes
[0887] To select natural high affinity TCRs, specifically
recognizing shared antigen MHC/peptide targets (SAT), the following
experimental steps are taken:
[0888] 1. Identification and isolation of MHC/peptide
target-reactive TCRs
[0889] 2. Production of engineered TCR T cells
[0890] 3. Verification of TCR specificity
[0891] Identification of MHC/Peptide Target-Reactive TCRs
[0892] T cells are isolated from blood, lymph nodes, or tumors of
patients. Patients are HLA-matched to SAT, and are selected based
on expression of target-harboring protein. T cells are then
enriched for SAT-specific T cells, e.g., by sorting SAT-MHC
tetramer binding cells or by sorting activated cells stimulated in
an in vitro co-culture of T cells and SAT-pulsed antigen presenting
cells.
[0893] SAT-relevant alpha-beta TCR dimers are identified by single
cell sequencing of TCRs of SAT-specific T cells. Alternatively,
bulk TCR sequencing of SAT-specific T cells is performed and
alpha-beta pairs with a high probability of matching are determined
using a TCR pairing method.
[0894] Alternatively or in addition, SAT-specific T cells can be
obtained through in vitro priming of naive T cells from healthy
donors. T cells obtained from PBMCs, lymph nodes, or cord blood are
repeatedly stimulated by SAT-pulsed antigen presenting cells to
prime differentiation of antigen-experienced T cells. TCRs are then
identified similarly as described above for SAT-specific T cells
from patients.
[0895] Production of Engineered TCR T Cells
[0896] TCR alpha and beta chain sequences are cloned into
appropriate constructs. TCR-autologous or heterologous bulk T cells
are transduced with the constructs to produce engineered TCR T
cells. These T cells are expanded in the presence of anti-CD3
antibodies and IL-2 cytokine for use in subsequent experiments. In
certain instances, native TCR is deleted or the inserted TCR is
modified to increase proper multimerization.
[0897] In Vitro Verification of TCR Specificity
[0898] First, T cells bearing engineered TCRs are screened for
target recognition using antigen presenting cells expressing the
appropriate MHC and pulsed with appropriate target(s).
[0899] TCRs identified in the first round of screening are then
tested for recognition of natural target. Lead TCRs are nominated
based on specific recognition of HLA-matched primary tumors and
tumor cell lines expressing SAT-harboring protein.
[0900] To assure specificity, lead TCRs are de-selected based on
off-target recognition. They are screened against a panel of HLA
matched and mismatched cell lines, covering multiple tissues and
organ types, and with HLA-matched and mismatched antigen presenting
cells pulsed with a panel of infectious disease antigens. TCRs with
specific and non-specific off-target recognition of self-antigens
or common non-self-antigens are de-selected.
Example 11: Identification of MHC/Peptide Target-Reactive TCRs
[0901] T cells are isolated from blood, lymph nodes, or tumors of
patients. Patients are HLA-matched to SAT, and are selected based
on expression of target-harboring protein. T cells are then
enriched for SAT-specific T cells, e.g., by sorting SAT-MHC
tetramer binding cells or by sorting activated cells stimulated in
an in vitro co-culture of T cells and SAT-pulsed antigen presenting
cells.
[0902] SAT-relevant alpha-beta TCR dimers are identified by single
cell sequencing of TCRs of SAT-specific T cells. Alternatively,
bulk TCR sequencing of SAT-specific T cells is performed and
alpha-beta pairs with a high probability of matching are determined
using a TCR pairing method.
[0903] Alternatively or in addition, SAT-specific T cells can be
obtained through in vitro priming of naive T cells from healthy
donors. T cells obtained from PBMCs, lymph nodes, or cord blood are
repeatedly stimulated by SAT-pulsed antigen presenting cells to
prime differentiation of antigen-experienced T cells. TCRs are then
identified similarly as described above for SAT-specific T cells
from patients.
Example 12: Production of Engineered TCR T Cells
[0904] TCR alpha and beta chain sequences are cloned into
appropriate constructs. TCR-autologous or heterologous bulk T cells
are transduced with the constructs to produce engineered TCR T
cells. These T cells are expanded in the presence of anti-CD3
antibodies and IL-2 cytokine for use in subsequent experiments. In
certain instances, native TCR is deleted or the inserted TCR is
modified to increase proper multimerization.
[0905] In Vitro Verification of TCR Specificity
[0906] First, T cells bearing engineered TCRs are screened for
target recognition using antigen presenting cells expressing the
appropriate MHC and pulsed with appropriate target(s).
[0907] TCRs identified in the first round of screening are then
tested for recognition of natural target. Lead TCRs are nominated
based on specific recognition of HLA-matched primary tumors and
tumor cell lines expressing SAT-harboring protein.
[0908] To assure specificity, lead TCRs are de-selected based on
off-target recognition. They are screened against a panel of HLA
matched and mismatched cell lines, covering multiple tissues and
organ types, and with HLA-matched and mismatched antigen presenting
cells pulsed with a panel of infectious disease antigens. TCRs with
specific and non-specific off-target recognition of self-antigens
or common non-self-antigens are de-selected.
Example 13: Identification of Monoclonal Antibodies (mAbs) that
Target MHC Class I Molecules Presenting Tumor Antigens Using Rabbit
B Cell Cloning Technologies
[0909] Potent and selective mAbs targeting human class I MHC
molecules presenting tumor antigens of interest are identified.
Soluble human pMHC molecules presenting human tumor antigens are
utilized for multiple mouse or rabbit immunizations followed by
screening of B cells derived from the immunized animals to identify
B cells that express mAbs that bind to target class I MHC
molecules. Sequences encoding the mAbs identified from the mouse or
rabbit screens will be cloned from the isolated B cells. The
recovered mAbs are then evaluated against a panel of irrelevant
pMHCs to identify lead mAbs that bind selectively to the target
pMHCs. Lead mAbs will be fully characterized to determine target
binding affinity and selectivity. Lead mAbs that demonstrate potent
and selective binding are humanized to generate full-length human
IgG monoclonal antibody (mAb) constructs. In addition, the lead
mAbs are incorporated into bi-specific mAb constructs and chimeric
antigen receptor (CAR) constructs that can be used to generate CAR
T-cells. Full-length bi-specifics or scFV-based bi-specifics can be
constructed.
[0910] Demonstrate Targeting of Human Tumor Cells In Vitro
[0911] Immunohistochemistry techniques are utilized to demonstrate
specific binding of lead antibodies to human tumor cells expressing
target pMHC molecules. T-cell lines transfected with CAR-T
constructs are incubated with human tumor cells to demonstrate
killing of tumor cells in vitro. Alternatively, tumor cells
expressing the target are incubated with bi-specific constructs
(encoding the ABP and an effector domain) and PBMCs or T cells.
[0912] In Vivo Proof-of-Concept
[0913] Lead antibody or CAR-T constructs are evaluated in vivo to
demonstrate directed tumor killing in humanized mouse tumor models.
Lead antibody or CAR-T constructs are evaluated in xenograft tumor
models engrafted with human PBMCs. Anti-tumor activity is measured
and compared to control constructs to demonstrate target-dependent
tumor killing.
[0914] Potent and selective ABPs that selectively target human
class I MHC molecules presenting tumor antigens will be identified
using phage display or B cell cloning technologies. The utility of
the ABPs will be demonstrated by showing that the ABPs mediated
tumor cell killing in vitro and in vivo when incorporated into
antibody or CAR-T cell constructs.
Example 14: Assessment of scFv-pHLA or Fab-pHLA Structures by
Hydrogen/Deuterium Exchange and Mass Spectrometry
[0915] Experimental Procedures
[0916] Hydrogen/Deuterium Exchange.
[0917] 20 .mu.M of HLA-peptide was incubated with a .about.3-fold
molar excess of scFv or Fab formatted proteins for 20 min at room
temperature (20-25.degree. C.) to generate complexes for the
exchange experiments. For the Apo (unbound) control, the
HLA-peptide was incubated with an equal volume of 50 mM NaCl, 20 mM
Tris pH 8.0. All subsequent reaction steps were performed at
4.degree. C. by an automated HDX PAL system controlled by Chronos
4.8.0 software (Leap Technologies, Morrisville, N.C.). 5 .mu.l of
protein complexes were diluted 10-fold into H20 or 50 mM NaCl, 20
mM Tris pH 8.0 (for the 0 min. control time-point) or the same
buffer made with D20 for 30s prior to quenching in 0.8 M guanidine
hydrochloride, 0.4% acetic acid (v/v), and 75 mM
tris(2-carboxyethyl) phosphine for 3 min. .about.50 pmol of
quenched protein complexes were transferred onto an immobilized
Protein XIII/Pepsin column (NovaBioAssays, Woburn, Mass.) for
integrated on-line protein digestion.
[0918] Liquid Chromatography, Mass Spectrometry, and HDX
Analysis
[0919] Chromatographic separation of peptides was carried out using
an UltiMate 3000 Basic Manual UHPLC System (ThermoFisher
Scientific, Waltham, Mass.), which contained a trap C18 column (5
.mu.M particle size and 2.1 mm diameter) and an analytical C18
column (1.9 .mu.M particle size and 1 mm diameter). Samples were
desalted with 10% acetonitrile, 0.05% trifluoroacetic acid or 10%
acetonitrile, 0.5% formic acid at a 40 .mu.l/min flow rate for 2
min and peptides were eluted at a 40 .mu.l/min flow rate with an
increasing concentration gradient of 95% acetonitrile with
trifluoro acetic acid or formic acid. Mass spectrometry was
performed with an Orbitrap Fusion Lumos mass spectrometer
(ThermoFisher, Waltham, Mass.) with the ESI source set at a
positive ion voltage of 3500-3800 V. Prior to performing
hydrogen-deuterium exchange experiments, peptide fragments of each
HLA-peptide complex were analyzed by data-dependent LC/MS/MS and
the data searched using PEAKS Studio (Bioinformatics Solutions
Inc., Waterloo, ON, Canada) with a peptide precursor mass tolerance
of 20 ppm and fragment ion mass tolerance of 0.2 Da. The HLA, 02M,
and target peptide sequences were searched, and false detection
rates identified using a decoy-database strategy. Peptides from the
hydrogen-deuterium experiments were detected by LC/MS and analyzed
by HDX Workbench (Omics Informatics, Honolulu, Hi.) with a
retention time window size of 0.22 min and a 7.0 ppm error
tolerance. High-resolution HD exchange data for selected peptides
were obtained by fragmenting the peptides by Electron Transfer
Dissociation (ETD) with a reaction time of 200 ms (G2) or 100 ms
(G10), using fluoranthene as the reagent anion. Peptide fragments
were analyzed by HDExaminer (Sierra Analytics) with a retention
time window size of 18s and a peptide m/z tolerance of 2 Da. Heat
maps of deuterium uptake differences were generated by Microsoft
Excel and mapped on to relevant protein crystallographic structures
using Pymol (Schrodinger, Cambridge, Mass.).
[0920] For the results below, amino acid numbering of the HLA alpha
helices is based on literal numbering of the mature protein, based
on the following: (1) removal of signal peptide, and (2) addition
of N-terminal methionine for bacterial expression. The HLA subtype
amino acid reference sequences and the beta-2 microglobulin amino
acid sequence are provided in Table 38.
[0921] Results
[0922] FIG. 21A shows an exemplary heatmap of the HLA portion of
the G8 HLA-PEPTIDE complex when incubated with scFv clone G8-P1H08,
visualized in its entirety using a consolidated perturbation
view.
[0923] FIG. 98 shows an example of high resolution data from scFv
clone G5-P1C12 plotted on crystal structure of HLA-B*35:01
(5xos.pdb; https://www.rcsb.org/structure/5XOS).
[0924] An example of the data from scFv G8-P1H08 plotted on the
crystal structure 1jf1.pdb, available at
http://www.rcsb.org/structure/1JF1, is shown in FIG. 21B.
[0925] An example of high-resolution HDX data from scFv G8-P1H08
plotted on a crystal structure of Fab clone G8-P1C11 complexed with
HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8"), is shown in FIG.
101.
[0926] FIG. 45A shows an exemplary heatmap of the HLA portion of
the G8 HLA-PEPTIDE complex when incubated with scFv clone G8-P1C11
(structure shown in FIG. 45B), visualized in its entirety using a
consolidated perturbation view.
[0927] An example of the data from scFv G8-P1C11 plotted on the
crystal structure described in Example 15 is shown in FIG. 45B.
[0928] FIG. 23A shows an exemplary heatmap of the HLA portion of
the G10 HLA-PEPTIDE complex when incubated with scFv clone
R3G10-P2G11, visualized in its entirety using a consolidated
perturbation view.
[0929] An example of the data from scFv R3G10-P2G11 plotted on a
crystal structure PDB5bs0 is shown in FIG. 23B. The crystal
structure, depicting a restricted peptide in the HLA binding cleft
formed by the .alpha.1 and .alpha.2 helices, can be found at URL
https://www.rcsb.org/structure/5bs0 (Raman et al).
[0930] An example of data from a second round of HDX studies, from
scFv-G10-P5A08, plotted on a crystal structure 5bs0.pdb is shown in
FIG. 23C. The crystal structure, depicting a restricted peptide in
the HLA binding cleft formed by the .alpha.1 and .alpha.2 helices,
can be found at URL https://www.rcsb.org/structure/5bs0 (Raman et
al).
[0931] To better compare the data across the ABPs tested for a
given HLA-PEPTIDE target, data for each ABP was exported, and a
heat map was generated in Excel. FIG. 22A shows resulting heat maps
from a first round of HDX experiments across the HLA .alpha.1 helix
for all ABPs tested for HLA-PEPTIDE target G8
(HLA-A*02:01_AIFPGAVPAA). FIG. 22B shows resulting heat maps across
the HLA .alpha.2 helix for all ABPs tested for HLA-PEPTIDE target
G8 (HLA-A*02:01_AIFPGAVPAA). FIG. 22C shows resulting heat maps
across the restricted peptide AIFPGAVPAA for all ABPs tested. The
heat maps from the first round of HDX data indicate positions 45-60
and 81-84 of the HLA protein (in the .alpha.1 helix) of HLA-PEPTIDE
target G8 (HLA-A*02:01_AIFPGAVPAA) as likely involved, directly or
indirectly, in determining the interaction between the HLA-PEPTIDE
target and G8-specific antibody-based ABPs.
[0932] FIG. 99 shows resulting color heat maps from high resolution
HDX experiments across the HLA .alpha.1 helix, the HLA .alpha.2
helix, and restricted peptide AIFPGAVPAA for all ABPs tested for
HLA-PEPTIDE target G8 (HLA-A*02:01_AIFPGAVPAA). FIG. 100 shows a
numerical representation of the color heat maps of FIG. 99. The
heat maps from the second round of HDX data indicate positions 46,
49, 55, 61, 74, 76, 77, 78, 81 and 84 of the HLA protein (in the
.alpha.1 helix) as likely involved, directly or indirectly, in
determining the interaction between the HLA-PEPTIDE target and
G8-specific antibody-based ABPs. The heat maps from the second
round of HDX data indicate positions 137, 138, 145, 147, 152-157 of
the HLA protein (in the .alpha.2 helix) as likely involved,
directly or indirectly, in determining the interaction between the
HLA-PEPTIDE target and G8-specific antibody-based ABPs. The heat
maps from the second round of HDX data indicate positions 5 and 6
of the restricted peptide AIFPGAVPAA as likely involved, directly
or indirectly, in determining the interaction between the
HLA-PEPTIDE target and G8-specific antibody-based ABPs.
[0933] FIG. 96 shows resulting color heat maps from high resolution
HDX experiments across the HLA .alpha.1 helix, the HLA .alpha.2
helix, and restricted peptide EVDPIGHVY for all ABPs tested for
HLA-PEPTIDE target G5 (HLA-B*35:01_EVDPIGHVY). FIG. 97 shows a
numerical representation of the color heat map of FIG. 96. These
heat maps indicate positions 50, 54, 55, 57, 61, 62, 74, 81, 82 and
85 of the HLA protein (in the .alpha.1 helix) as likely involved,
directly or indirectly, in determining the interaction between the
HLA-PEPTIDE target and G5-specific antibody-based ABPs. These heat
maps indicate positions 147 and 148 of the HLA protein (in the
.alpha.2 helix) as likely involved, directly or indirectly, in
determining the interaction between the HLA-PEPTIDE target and
G5-specific antibody-based ABPs.
[0934] FIG. 24A shows resulting heat maps from a first round of HDX
experiments across the HLA .alpha.1 helix for all ABPs tested for
HLA-PEPTIDE target G10 (HLA-A*01:01_ASSLPTTMNY). FIG. 24B shows
resulting heat maps from a first round of HDX experiments across
the HLA .alpha.2 helix for all ABPs tested for HLA-PEPTIDE target
G10 (HLA-A*01:01_ASSLPTTMNY). FIG. 24C shows resulting heat maps
from a first round of HDX experiments across the restricted peptide
ASSLPTTMNY for all ABPs tested. FIG. 92 shows resulting heat maps
from a second round of HDX experiments across the HLA .alpha.1
helix, the HLA .alpha.2 helix, and the restricted peptide
ASSLPTTMNY for all ABPs tested. Taken together, the heat maps
indicate positions 49-56 and/or 59-66 of the HLA protein (in the
.alpha.1 helix), as well as positions 136-147 and 157-160 of the
.alpha.2 helix of the HLA protein, as likely involved, directly or
indirectly, in determining the interaction between the HLA-PEPTIDE
target and G10-specific antibody-based ABPs. In particular, all of
the ABPs tested decreased solvent accessibility of positions 52-54
of the HLA .alpha.1 helix.
[0935] An example of the data from scFv G2-P1G07 plotted on a
crystal structure PDB 5bs0 is shown in FIG. 77. The crystal
structure can be found at URL https://www.rcsb.org/structure/5bs0
(Raman et al). Areas not covered with MS data are shown in black
and those with the greatest decrease in D exchange (indicating a
binding site for the ABP) is circled. For clarity, only the binding
groove and helices are shown.
[0936] An exemplary heatmap for scFv clone G2-P1G07 visualized in
its entirety using a consolidated perturbation view is shown in
FIG. 78.
[0937] An example of the data from scFv G2-P2C11 plotted on a
crystal structure PDB 5bs0 is shown in FIG. 94.
[0938] FIG. 95 shows high resolution HDX data plotted on a crystal
structure PDB 5bs0. Data for G2 bound to four different scFvs were
obtained by fragmenting peptides by Electron Transfer Dissociation
(ETD) as described in the Experimental Procedures.
[0939] To better compare the data across the ABPs tested for a
given HLA-PEPTIDE target, data for each ABP was exported, and a
heat map was generated in Excel. Resulting heat maps are shown in
FIG. 79 showing a heat map across the .alpha.1 helix (top) and
across the .alpha.2 helix (bottom). FIG. 80 shows a heat map for
all ABPs tested for A*01:01_NTDNNLAVY, across restricted peptide
residues 1-9. Heat maps from a second (higher resolution) round of
HDX data are shown in FIG. 93. Taken together, the heat maps
elucidated regions of reduced solvent accessibility in the HLA
alpha subunits that bind and display the target peptide. Many of
these regions were shared across multiple A*01:01_NTDNNLAVY
specific ABPs. The two regions which most commonly exhibited
decreased solvent accessibility include A70-Y85 of the alpha 1
helix, and/or positions A140-Y160 of the alpha 2 helix, with all
ABPs shielding R157-Y160 of the helix. Taken together, the heat
maps also indicate HLA-PEPTIDE/ABP interactions that decrease
solvent accessibility across positions 3-9 of the restricted
peptide. The effect was increasingly pronounced towards the
C-terminal direction. This pattern was consistent for 14 of the 15
antibodies examined, with positions 6-9 invariably being shielded
by the presence of the ABPs. All clone entries in the HDX heat maps
are scFv formats unless otherwise noted.
[0940] G7 (A*02:01_LLASSILCA) scFv clones P2E09 and P3A09 were
assessed by HDX-MS according to the methods described above.
Solvent accessibility was decreased in a region of the HLA-A*02:01
alpha 1 helix corresponding to positions 49-85, with an overlap of
G57-K67 (data not shown). Solvent accessibility was also decreased
in a region of the alpha 2 helix from positions 136-157, with an
overlap between positions 144 and 152. Taken together, these leads
cover a broad footprint on HLA.
Example 15: Assessment of Fab-PHLA Structures by
Crystallography
[0941] Materials and Methods
[0942] Complex Purification and Crystal Screening
[0943] Fab fragments corresponding to, e.g., HLA-PEPTIDE target G8
(A*02:01_AIFPGAVPAA) were concentrated to reach 5 mg/mL (100M)
before addition of its corresponding HLA-MHC (1:1 molar ratio) and
incubated for 30 minutes at 4.degree. C. The mixture was then
injected on size exclusion chromatography column (S200 16/60)
equilibrated in 1.times.PBS buffer for complex purification.
Fractions containing both Fab and HLA and with an elution volume
consistent with a complex of .about.94 kDa were pooled and
concentrated to 10-12 mg/mL (1AU=1 mg/mL). Each purified complex
was screened for crystallization conditions using commercial
screens: PEGIon (Hampton research), JCSG+(Molecular Dimensions) and
JBS Screen 3 and 4 (Jena Biosciences). The choice of the kits was
driven by the characteristic of known crystal conditions of HLA-Fab
complexes that are mainly based on the use of PEG3350 or PEG4000 as
precipitant. 3 to 4 weeks after screen, diffraction suitable
crystals appeared for HLA-Fab combinations in several
crystallization conditions (Table 24). The protein nature of the
crystals was checked by UV. Crystals were transferred into a
cryoprotectant solution (crystallization solution supplemented with
25% Glycerol) and flash frozen in liquid nitrogen.
[0944] Data Collection and Processing
[0945] Diffraction data was collected on the Proxima 2A beamline at
SOLEIL synchrotron (Gif sur Yvette, France). Data processing and
scaling was performed using XDS (1). Molecular replacement was
performed using MolRep and Arp/Warp from the CCP4 suite (2) using
PDB 5E61 for HLA (100% sequence identity) and 5AZE (90% sequence
identity with VH) and 5115 (97% sequence identity with VL) for Fab
as entry models. Refinement was performed using Buster TNT
(GlobalPhasing, Inc) and manual model modifications in Coot (CCP4
suite).
[0946] Complex Purification
[0947] Combinations produced a good separation between the
individual protein peak and the formed complex peak (FIG. 28A).
Increasing incubation time to 16 hours (overnight) did not change
the ratio of complex formed (.about.50% of the protein is present
in complex and 50% as free proteins). Peak analysis by SDS PAGE
under reducing conditions showed the presence of both Fab chains
(30 kDa), HLA heavy chain (.about.35 kDa), and HLA light chain
(BLM, 10 kDa) in the pooled fractions (FIG. 28).
[0948] Crystallization and Data Collection
Complex pooled fractions were concentrated and screened. After 3-4
weeks crystals appeared for some of the HLA-Fab combinations. A
summary of the crystallography conditions for the
A*02:01_AIFPGAVPAA-G8-P1C11 Fab complex and resulting crystal
formation is shown in Table 24.
TABLE-US-00043 TABLE 24 Crystallography conditions Com- Crystals
mercial Obtained Kit Experimental Conditions (Y/N) JBS 20% PEG4000,
200 mM Magnesium sulfate, No 10% glycerol (GOL) JBS 20% PEG4000,
200 mM Magnesium sulfate, Yes 5% 2-Propanol JBS 20% w/v
Polyethylene glycol 4,000 10% w/v No 2-Propanol, 100 mM HEPES; pH
7.5 JCSG 20% (w/v) PEG 3350 200 mM Ammonium No chloride JCSG 30%
(w/v) PEG 2000 MME 100 mM No Potassium thiocyanate JCSG 25% (w/v)
PEG 3350 100 mM Bis-Tris/ Yes Hydrochloric acid pH 5.5 (integrated
into P1 Space group) JCSG 30% v/v Jeffamine .RTM. M-600, 0.1M Yes
HEPES pH 7.0 JCSG 25% (w/v) PEG 3350 100 mM Bis-Tris/ No
Hydrochloric acid pH 5.5, 200 mM Lithium sulfate PEGion 0.2M
Ammonium tartrate dibasic pH 7.0, 20% Yes w/v Polyethylene glycol
3,350 (integrated into P1 Space group) PEGion 2% v/v TacsimateTM pH
6.0 0.1M BIS-TRIS No pH 6.5 20% PEG3350 PEGion 1% w/v Tryptone
0.001M Sodium azide, No 0.05M HEPES sodium pH 7.0, 20% w/v
Polyethylene glycol 3,350
[0949] Out of the tested conditions, four yielded crystals. Two
yielded crystals which diffracted well (1.7 to 2.0 .ANG.
resolution) and were integrated into a P1 space group (Table 24).
Structure resolution was possible by combining molecular
replacement (MolRep) and software automated model building using
Arp/Warp.
[0950] An exemplary crystal of a complex comprising Fab clone
G8-P1C11 and HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8") is shown
in FIG. 29. This crystal was grown using the commercial screen
JCSG, using 25% (w/v) PEG 3350 100 mM Bis-Tris/Hydrochloric acid pH
5.5. This crystal was used to generate the structural data
below.
[0951] Structural Analysis
[0952] The overall structure of a complex formed by binding of Fab
clone G8-P1C11 to HLA-PEPTIDE target A*02:01_AIFPGAVPAA ("G8") is
shown in FIG. 30. The individual proteins are represented as
surfaces. The interface area between the HLA and the VH and VL is
747 .ANG..sup.2 and 285 .ANG..sup.2, respectively.
[0953] During refinement electron density region corresponding to
the peptide was clearly visible and allowed peptide side chain
unambiguous positioning (FIG. 31) with the provided 10 residue
peptide sequence AIFPGAVPAA. All areas relevant to interaction
interfaces are refined; however, some refinement is still required
in antibody constant regions.
[0954] Coding of monomers in the complex, which is referred to in
the following data, is provided in Table 25 below.
TABLE-US-00044 TABLE 25 monomer coding used in crystal analysis
Monomer Monomer Code (ID) HLA heavy chain (.alpha.1, .alpha.2,
.alpha.3) A HLA .beta.2 microglobulin (light chain) B Restricted
peptide I Fab heavy chain (VH-CH1) C Fab light chain (VL-CL) D
[0955] HLA-Peptide Interaction
[0956] The restricted peptide AIFPGAVPAA is mainly buried in the
HLA A*02:01 binding pocket with the residues P4G5A6 protruding
towards the Fab. The interaction surface between the peptide and
the HLA is 926 .ANG..sup.2 and represents 76% of the total peptide
solvent accessible surface (1215 .ANG..sup.2). The binding of the
peptide to the HLA involves 9 hydrogen bonds and van der Waals
interactions (FIG. 32) and yields a binding energy of -16.4
kcal/mol.
[0957] A list of hydrogen interactions is shown in Table 26,
below.
TABLE-US-00045 TABLE 26 Hydrogen bond interactions between
restricted peptide and HLA. Distance Peptide (Angstroms) HLA I:ALA
1[ N ] 2.72 A:TYR 172[ OH ] I:ALA 1[ N ] 2.86 A:TYR 8[ OH ] I:ILE
2[ N ] 2.81 A:GLU 64[ OE1 ] I:ILE 2[ N ] 3.71 A:TYR 8[ OH ] I:PHE
3[ N ] 2.94 A:TYR 100[ OH ] I:ALA 1[ O ] 2.67 ] A:TYR 160[ OH I:PRO
8[ O ] 2.93 A:ARG 98[ NH2 ] I:PRO 8[ O ] 2.89 A:ARG 98[ NH1 ] I:ALA
9[ O ] 2.71 A:TRP 148[ NE1 ] I:ALA 1[ N ] 2.72 A:TYR 172[ OH ]
[0958] A complete interface summary of the HLA and restricted
peptide is shown in FIG. 37.
[0959] A complete list of the interacting residues from the
restricted peptide and HLA is shown in FIG. 38.
[0960] Fab-Restricted Peptide Interactions
[0961] As most of the peptide is buried in the binding pocket of
the HLA, only part of it available for interactions with the Fab
chains. This is confirmed by the observation that 76% of the
solvent accessible area of the peptide is occupied by its
interaction with the HLA. Interaction surface between the peptide
and the heavy chain and the light chain of the Fab is 114.3 and
113.9 .ANG..sup.2 respectively. This corresponds to 18% of the
total peptide solvent accessible area. PISA analysis showed that
only two hydrogen bonds are involved in the interaction between the
Fab and the peptide: hydroxyl group of Tyr32 from the light chain
interacts with the backbone carbonyl of Gly5 of the peptide and the
Tyr100A backbone amide interacting with the backbone carbonyl group
of Pro4 of the peptide (See Table 27 for a list of the hydrogen
interactions, below).
TABLE-US-00046 TABLE 27 Fab/restricted peptide H bond interactions
Peptide Distance (A) Fab I:PRO 4[ O ] 3.0 C:TYR 100A[ OH ] (VH)
I:GLY 5[ O ] 3.7 D:TYR 32[ OH ] (VL)
[0962] The recognition mode of the Fab towards the restricted
peptide is mainly through hydrophobic interactions and hydrogen
bonds involving solvent molecules (FIGS. 33 and 34). The binding
energy of the interaction between the Fab and restricted peptide is
-2.0 and -1.9 kcal/mol with the VH and VL chains respectively.
[0963] A complete interface summary of the Fab VH chain and
restricted peptide, and a complete list of the interacting residues
from the Fab VH chain and restricted peptide, is shown in FIG.
39.
[0964] A complete interface summary of the Fab VL chain and
restricted peptide, and a complete list of the interacting residues
from the Fab VL chain and restricted peptide, is shown in FIG.
40.
[0965] Fab-HLA Interactions
[0966] The Fab and the HLA moieties interacts extensively as shown
by interface area between the HLA and the Fab with a total of 1032
.ANG..sup.2. The interaction between the HLA and the VH chain is
composed of hydrophobic interactions, 6 H bonds and 3 salt bridges
(FIG. 35, interaction between VH and HLA; and FIG. 36, interaction
between VL and HLA). This interaction represents the major
interaction are with 747 .ANG..sup.2 (72% of the total contact
area).
[0967] A table of the hydrogen bond contacts between the VH chain
of the Fab and the HLA protein is shown below.
TABLE-US-00047 TABLE 28 hydrogen bond contacts between VH and HLA.
Fab VH Distance HLA C:SER 31[ OG ] 2.71 A:THR 164[ OG1 ] C:TYR
100A[ OH ] 2.55 A:THR 164[ OG1 ] C:SER 31[ N ] 3.17 A:GLU 167[ OE1
] C:SER 30[ N ] 2.86 A:GLU 167[ OE2 ] C:TYR 32[ OH ] 2.80 A:LYS 67[
NZ ] C:TYR 98[ O ] 2.94 A:ARG 66[ NH2 ] C:ASP 100[ OD1 ] 2.88 A:ARG
66[ NH1 ]
[0968] A table of the salt bridge contacts between the VH chain of
the Fab and the HLA protein is shown below.
TABLE-US-00048 TABLE 29 salt bridge contacts between VH and HLA.
Fab VH Distance HLA C:ASP 100[ OD1 ] 2.88 A:ARG 66[ NH1 ] C:ASP
100[ OD1 ] 3.39 A:ARG 66[ NH2 ] C:ASP 100[ OD2 ] 3.40 A:ARG 66[ NH1
]
[0969] A complete interface summary of the Fab VH chain HLA protein
is shown in FIG. 41.
[0970] A complete list of the interacting residues from the Fab VH
chain and HLA protein is shown in FIG. 42.
[0971] A table of the hydrogen bond contacts between the VL chain
of the Fab and the HLA protein is shown in Table 30 below.
TABLE-US-00049 TABLE 30 hydrogen bonds between VL and HLA. Fab VL
Distance HLA D:ILE 94[ N ] 3.56 A:ALA 151[ O ] D:SER 30[ OG ] 2.84
A:GLN 73[ NE2 ] D:ILE 94[ O ] 3.00 A:HIS 152[ ND1 ]
[0972] A complete interface summary of the Fab VL chain HLA protein
is shown in FIG. 43.
[0973] A complete list of the interacting residues from the Fab VL
chain and HLA protein is shown in FIG. 44.
Example 16: Identification of Predicted HLA-PEPTIDE Complexes
[0974] We identified cancer specific HLA-peptide targets using
three computational steps: First, we identified genes that are not
generally expressed in most normal tissues using data available
through the Genotype-Tissue Expression (GTEx) Project [1]. We then
identified which of those genes are aberrantly expressed in cancer
samples using data from The Cancer Genome Atlas (TCGA) Research
Network: http://cancergenome.nih.gov/. In these genes, we
identified which peptides are likely to be presented as cell
surface antigens by MHC Class I proteins using a deep learning
model trained on HLA presented peptides sequenced by MS/MS, as
described in international patent application no.
PCT/US2016/067159, herein incorporated by reference, in its
entirety, for all purposes.
[0975] To identify genes that are not usually expressed in normal
tissues, we obtained aggregated gene expression data from the
Genotype-Tissue Expression (GTEx) Project (version V6p). This
dataset comprised 8,555 post-mortem samples from over 50 tissue
types. Expression was measured using RNA-Seq and computationally
processed according to the GTEx standard pipeline
(https://www.gtexportal.org/home/documentationPage). For the
purposes of this analysis, genes were considered not expressed in
normal tissues if they were found not to be expressed in any
tissues in GTEx or were only expressed in one or more of testis,
minor salivary gland, and the endocervix (i.e., immune privileged
or non-essential tissues). We also restricted our search to only
include protein coding genes. Because GTEx and TCGA use different
annotations of the human genome in their computational analyses, we
excluded genes which we could not map between the two datasets
using standard techniques such as ENCODE mappings.
[0976] We sought to define criteria to excluded genes that were
expressed in normal tissue that was strict to ensure tumor
specificity, but would not exclude non-zero measurements arising
from sporadic, low level transcription or potential artifacts such
as read misalignment. Therefore, we designated a gene to be not
normally expressed in a non-immune privileged or essential tissue
if its median expression across GTEx samples was less than 0.5 RPKM
(Reads Per Kilobase of transcript per Million mapped reads), and it
was never expressed with greater than 10 RPKM, and it was expressed
at 5 RPKM in no more than two samples across all essential tissue
samples. To exclude genes which were potentially expressed but
could not be measured by RNA-Seq using the GTEX analysis pipeline,
we also excluded genes which were measured at 0 RPKM in all
samples. These criteria left us with a set of protein coding genes
that did not appear to be expressed in most normal tissues.
[0977] We next sought to identify which of these genes are
aberrantly expressed in tumors. We examined 11,093 samples
available from TCGA (Data Release 6.0). We considered a gene
expressed if it was observed at expression of at least 5 FPKM
(Fragments Per Kilobase of transcript per Million mapped reads) in
at least 5 samples. Because one fragment usually consists of two
mapped reads, 5 FPKM equals approximately 10 RPKM.
[0978] While the GTEx data spans a broad range of tissue types, it
does not include all cell types that are present in the human body.
We therefore further examined the list for the gene's biological
function category using the DAVID v 6.8 [2] and used this analysis,
along with literature review, to filter the gene list further. We
removed genes likely to be expressed in immune cells (e.g.,
interferon family genes), eye-related genes (e.g., retina in the
FANTOM5 dataset http://www.proteinatlas.org), genes expressed in
the mouth and nose (e.g. olfactory genes and taste receptors), and
genes related to the circadian cycle. We also excluded genes that
are part of large gene families, including histone genes, because
their expression is difficult to accurately assess with RNA
Sequencing due to sequence homology.
[0979] We then examined the distribution of the expression of the
remaining genes across the TCGA samples. When we examined the known
Cancer Testis Antigens (CTAs), e.g., the MAGE family of genes, we
observed that the expression of these genes in log space was
generally characterized by a bimodal distribution across samples in
the TCGA. This distribution included a left mode around a lower
expression value and a right mode (or thick tail) at a higher
expression level. This expression pattern is consistent with a
biological model in which some minimal expression is detected at
baseline in all samples and higher expression of the gene is
observed in a subset of tumors experiencing epigenetic
dysregulation. We reviewed the distribution of expression of each
gene across TCGA samples and discarded those where we observed only
a unimodal distribution with no significant right-hand tail, as
this distribution may (as a non-limiting example) more likely
characterize genes that have a low baseline of expression in normal
tissues.
[0980] This left us with a remaining gene list of >630 genes
that was highly enriched for genes involved in testis-specific
biological processes and development. Because many of these genes
produce different isoforms, these genes mapped to >1,200
proteins using the UNIPROT mapping service. In addition to the
genes that met our strict computational criteria, we added several
genes that have previously been identified in the scientific
literature as cancer testes antigens.
[0981] To identify the peptides that are likely to be presented as
cell surface antigens by MHC Class I proteins, we used a sliding
window to parse each of these proteins into its constituent 8-11
amino acid sequences. We processed these peptides and their
flanking sequences with the HLA peptide presentation deep learning
model to calculate the likelihood of presentation of each peptide
at expression levels between five TPM, which approximately
corresponds to one transcript per cell [3], to 200 TPM (i.e., a
high level of expression). We considered a peptide a putative
HLA-PEPTIDE target if its probability of presentation calculated by
our model was greater than 0.1 in 10 or more patients in the TCGA
dataset with expression 5 TPM or greater.
[0982] The results are shown in Table A1. From this example, there
are >1,800 HLA-PEPTIDE targets across .about.400 genes and 25
analyzed HLA alleles. For clarity, each HLA-PEPTIDE was assigned a
target number in Table A1. For example, HLA-PEPTIDE target 1 is
HLA-A*01:01_EVDPIGHLY, HLA-PEPTIDE target 2 is
HLA-A*29:02_FVQENYLEY, and so forth.
[0983] Collectively, this list of HLA-PEPTIDE targets is expected
to be a significant contribution to the state of knowledge of
cancer specific targets. In summary, the example provides a large
set of tumor-specific HLA-PEPTIDEs that can be pursued as candidate
targets for ABP research and development.
REFERENCES
[0984] 1. Consortium, G. T., The Genotype-Tissue Expression
(GTEx)project. Nat Genet, 2013. 45(6): p. 580-5. [0985] 2. Huang
da, W., B. T. Sherman, and R. A. Lempicki, Systematic and
integrative analysis of large gene lists using DAVID bioinformatics
resources. Nat Protoc, 2009. 4(1): p. 44-57. [0986] 3. Shapiro, E.,
T. Biezuner, and S. Linnarsson, Single-cell sequencing-based
technologies will revolutionize whole-organism science. Nat Rev
Genet, 2013. 14(9): p. 618-30.
Example 17: Initial Validation of Predicted HLA-PEPTIDE
Complexes
[0987] As an initial assessment to validate the predicted
HLA-PEPTIDE targets arising from the above described approach, we
evaluated public databases and selected literature for reports of
these targets as having been previously identified by various assay
techniques, including HLA binding affinity measurements, HLA
peptide mass-spectrometry, as well as measures of T cell responses.
Two comprehensive databases containing assay result annotations for
HLA-PEPTIDE pairs were used: IEDB (Vita et al., 2015) and Tantigen
(Olsen et al., 2017). We determined that 19 (15 unique across
genes) of the computationally predicted targets were previously
reported in the databases, many in genes (e.g., cancer testis
antigens) that have long been the subject of study in cancer
immunology. See Table B.
TABLE-US-00050 TABLE B Found in Protein IEDB or IEDB Tantigen Name
HLA-PEPTIDE Tantigen Status Status MAGA3 HLA-A*01:01_EVDPIGHLY TRUE
Found Found MAGA3 HLA-A*29:02_FVQENYLEY TRUE Found Not found MAGA3
HLA-A*29:02_LVHFLLLKY TRUE Found Not found MAGA3
HLA-B*44:03_MEVDPIGHLY TRUE Not found Found MAGA6
HLA-A*29:02_FVQENYLEY TRUE Found Not found MAGA6
HLA-A*29:02_LVHFLLLKY TRUE Found Not found MAGA4
HLA-A*01:01_EVDPASNTY TRUE Not found Found MAGA1
HLA-A*02:01_KVLEYVIKV TRUE Found Found MAGAC HLA-A*29:02_LVHFLLLKY
TRUE Found Not found MAGAC HLA-A*29:02_LVQENYLEY TRUE Found Not
found SSX1 HLA-C*04:01_AFDDIATYF TRUE Found Not found MAGA4
HLA-A*29:02_WVQENYLEY TRUE Found Not found MAGB2
HLA-A*02:01_GVYDGEEHSV TRUE Found Not found MAGA1
HLA-A*03:01_SLFRAVITK TRUE Found Found MAGA4 HLA-A*11:01_ALAETSYVK
TRUE Found Not found SAGE1 HLA-A*24:02_LYATVIHDI TRUE Not found
Found PASD1 HLA-A*02:01_QLLDGFMITL TRUE Found Not found MAGA8
HLA-A*29:02_WVQENYLEY TRUE Found Not found MAGAC
HLA-A*29:02_STLPTTINY TRUE Found Not found
[0988] Additional limited literature review was carried out for
peptides not found in the above public databases. The following
peptides were identified, as shown in Table C:
TABLE-US-00051 TABLE C HLA/peptide known HLA/peptide known status
in HLA Protein status IEDB or literature (preliminary) if
allele/peptide complex Name Tantig 2017 not in IEDB or Tantigen
HLA-A*01:01_NTDNNLAVY KKLC1 Not known WO 2017/089756 A1 (Stevanovi
etal., 2017) HLA-B*35:01_YPAPLESLDY PRA10 Not known WO2008118017 A2
HLA-A*11:01_ATLENLLSH PRAM4 Not known WO2008118017 A2
HLA-B*51:01_DALLAQKV PRA12 Not known WO2008118017 A2
HLA-B*44:03_SESDLKHLSW PRA12 Not known WO2008118017 A2
HLA-A*11:01_ATLENLLSH PRAM9 Not known WO2008118017 A2
HLA-A*02:07_TLDEYLTYL PRAM9 Not known WO2008118017 A2
[0989] One notable example from Table C was KKLC1
HLA-A*01:01_NTDNNLAVY Kita-kyushu lung cancer antigen-1 (KK-LC-1;
CT83) is a cancer testis antigen (CTA) that has been shown to be
widely expressed in many different cancer types. It was originally
discovered based on a cloned CTL to KK-LC-1 peptide
76-84--RQKRILVNL (Fukuyama et al., 2006). More recently Stevanovid
et al., 2017 revealed another peptide from KK-LC-1 recognized by a
CTL in a patient with cervical cancer, the predicted peptide
KK-LC-1 52-60 NTDNNLAVY The corresponding TCR for this CTL is now
listed on the NIH website
https://www.ott.nih.gov/technology/e-153-2016/and the peptide is
listed in WO 2017/089756 A1, herein incorporated by reference, in
its entirety, for all purposes.
[0990] This example highlights the expected value of predicted
HLA-PEPTIDE targets in Table A: Although no information on which
CTA HLA-PEPTIDE targets were previously known was incorporated in
the prediction, the analysis yielded many targets that were
described in the literature, indicating that many of the novel
targets can likewise be validated experimentally and ultimately
serve as targets for one or more ABPs.
REFERENCES
[0991] Fukuyama, T., Hanagiri, T., Takenoyama, M., Ichiki, Y,
Mizukami, M., So, T., Sugaya, M., So, T., Sugio, K., and Yasumoto,
K. (2006). Identification of a new cancer/germline gene, KK-LC-1,
encoding an antigen recognized by autologous CTL induced on human
lung adenocarcinoma. Cancer Res. 66, 4922-4928. [0992] Olsen, L.
R., Tongchusak, S., Lin, H., Reinherz, E. L., Brusic, V., and
Zhang, G. L. (2017). TANTIGEN: a comprehensive database of tumor T
cell antigens. Cancer Immunol. Immunother. CII 66, 731-735. [0993]
Stevanovi , S., Pasetto, A., Helman, S. R., Gartner, J. J.,
Prickett, T. D., Howie, B., Robins, H. S., Robbins, P. F.,
Klebanoff, C. A., Rosenberg, S. A., et al. (2017). Landscape of
immunogenic tumor antigens in successful immunotherapy of virally
induced epithelial cancer. Science 356, 200-205. [0994] Vita, R.,
Overton, J. A., Greenbaum, J. A., Ponomarenko, J., Clark, J. D.,
Cantrell, J. R., Wheeler, D. K., Gabbard, J. L., Hix, D., Sette,
A., et al. (2015). The immune epitope database (IEDB) 3.0. Nucleic
Acids Res. 43, D405-412.
Example 18: Identification of Predicted HLA-PEPTIDE Complexes
[0995] Next, HLA-peptide targets from proteins of seven genes were
identified: AFP, KKLC-1, MAGE-A4, MAGE-A10, MART-1, NY-ESO-1, and
WT1.
[0996] To identify peptides that are likely to be presented as cell
surface antigens by MHC Class I proteins, a sliding window was used
to parse each of these proteins into its constituent 8-11 amino
acid sequences. These peptides and their flanking sequences were
then processed with the HLA peptide presentation deep learning
model (see PCT/US2016/067159 and Example 16 above) to calculate the
likelihood of presentation of each peptide at an expression level
of 100 TPM (high expression) for each of 64 Class I HLA types.
Potential modeling artifacts were removed that could give stronger
scores to certain HLAs due to training data biases by quantile
normalizing model scores for each HLA so that each HLA present
scores from the same distribution. In the normalization, the seven
target genes as well as 50 randomly selected genes were included to
control for HLA allele sequence preferences. A gene was considered
likely to be presented if the model normalized score was higher
than 0.00075, which was chosen based on the presentation scores of
peptides known to be presented in the literature.
[0997] The results are shown in Table A2. Target numbers were
assigned to each HLA-PEPTIDE target as described in Example 16.
Example 19: Identification of Antibodies or Antigen-Binding
Fragments Thereof that Bind HLA-PEPTIDE Complexes
[0998] Overview
[0999] The following exemplification demonstrates that antibodies
(Abs) can be identified that recognize tumor-specific
HLA-restricted peptides. The overall epitope that is recognized by
such Abs generally comprises a composite surface of both the
peptide as well as the HLA protein presenting that particular
peptide. Abs that recognize HLA complexes in a peptide-specific
manner are often referred to as T cell receptor (TCR)-like Abs or
TCR-mimetic Abs. The HLA-PEPTIDE target antigens that were selected
for antibody discovery are HLA-A*01:01_NTDNNLAVY (Target 33 in
Table A1 designated as "G2") and HLA-A*02:01_LLASSILCA (Target 6427
in Table A2, designated as "G7"). Cell surface presentation of
these HLA-PEPTIDE antigens was confirmed by mass spectrometry
analysis of HLA complexes obtained from tumor samples, as described
in Example 2.
[1000] Generation of HLA-PEPTIDE Target Complexes and Counterscreen
Peptide-HLA Complexes, and Stability Analysis
[1001] The HLA-PEPTIDE targets G2 and G7, as well as counterscreen
negative control peptide-HLAs, were produced recombinantly using
conditional ligands for HLA molecules using established methods. In
all, 18 counterscreen HLA-peptides were generated for each of the
G2 and G7 targets.
[1002] Overall Design of Phage Library Screening
[1003] The highly diverse SuperHuman 2.0 synthetic naive scFv
library from Distributed Bio Inc (7.6e10 total diversity on
ultra-stable and diverse VH/VL scaffolds) was used for phage
display. The phage library was initially depleted with 18 pooled
negative pHLA complexes (the "complete pool") followed by three to
four rounds of bead-based phage panning with the target pHLA
complex using established protocols to identify scFv binders to
HLA-PEPTIDE targets G2 and G7, respectively. The phage titer was
determined at every round of panning to establish removal of
non-binding phage. Phage ELISA results are shown in FIGS. 70A and
70B. There was an enrichment of bound phage in later rounds of
panning for each of the G2 and G7 targets. The output phage
supernatant was also tested for target binding by ELISA.
[1004] The design of target screen 1 for the G2 target is shown in
FIG. 64. Similarly, the design of target screen 2 for the G7 target
is shown in FIG. 67. Briefly, for each target, three "minipool"
counterscreen peptides were selected for their ability to bind the
same HLA allele as the target and also to have significantly
different ABP-facing features such as charge, bulk, aromatic, or
hydrophobic residues. See FIG. 65A for G2 and FIG. 69A for G7. In
addition, additional counterscreen peptide-HLA complexes, featuring
distinct restricted peptide sequences and different HLA alleles
were generated. The 15 additional counterscreen HLA-peptides plus
the three "minipool" HLA-peptides formed a "complete pool" of 18
total counterscreen HLA-peptide complexes.
[1005] Generation of Peptide-HLA Complexes
[1006] .alpha.-, and .beta.2 microglobulin chain of various human
leukocyte antigens (HLA) were expressed separately in BL21
competent E. Coli cells (New England Biolabs) using established
procedures (Garboczi, Hung, & Wiley, 1992). Following
auto-induction, cells were lysed via sonication in Bugbuster.RTM.
plus benzonase protein extraction reagent (Novagen). The resulting
inclusion bodies were washed and sonicated in wash buffer with and
without 0.5% Triton X-100 (50 mM Tris, 100 mM NaCl, 1 mM EDTA).
After the final centrifugation, inclusion pellets were dissolved in
urea solution (8 M urea, 25 mM MES, 10 mM EDTA, 0.1 mM DTT, pH
6.0). Bradford assay (Biorad) was used to quantify the
concentration and the inclusion bodies were stored at -80.degree.
C.
[1007] HLA complexes were obtained by refolding of recombinantly
produced subunits and a synthetically obtained peptide using
established procedures. (Garboczi et al., 1992). Briefly, the
purified .alpha. and .beta.2 microglobulin chains were refolded in
refold buffer (100 mM Tris pH 8.0, 400 mM L-Arginine HCl, 2 mM
EDTA, 50 mM oxidized glutathione, 5 mM reduced glutathione,
protease inhibitor tablet) with the restricted peptide of choice.
In some experiments, the restricted peptide of choice was a
conditional ligand peptide, which is cleavable upon exposure to a
conditional stimulus. In some experiments, the restricted peptide
of choice was the G2 or G7 target peptide, or counterscreen
peptide. The refold solution was concentrated with a Vivaflow 50 or
50R crossflow cassette (Sartorius Stedim). Three rounds of dialyses
in 20 mM Tris pH 8.0 were performed for at least 8 hours each. For
the antibody screening and functional assays, the refolded HLA was
enzymatically biotinylated using BirA biotin ligase (Avidity).
Refolded protein complexes were purified using a HiPrep (16/60
Sephacryl S200) size exclusion column attached to an Akta FPLC
system. Biotinylation was confirmed in a streptavidin gel-shift
assay under non-reducing conditions by incubating the refolded
protein with an excess of streptavidin at room temperature for 15
minutes prior to SDS-PAGE. The resulting peptide-HLA complexes were
aliquoted and stored at -80.degree. C.
[1008] Stability Analysis of the Peptide-HLA Complexes
[1009] HLA-peptide stability was assessed by conditional ligand
peptide exchange and stability ELISA assay. Briefly, conditional
ligand-HLA complexes were subjected to .+-.conditional stimulus in
the presence or absence of the counterscreen or test peptides.
Exposure to the conditional stimulus cleaves the conditional ligand
from the HLA complex, resulting in dissociation of the HLA complex.
If the counterscreen or test peptide stably binds the
.alpha.1/.alpha.2 groove of the HLA complex, it "rescues" the HLA
complex from disassociation.
[1010] The HLA stability ELISA was performed using established
procedures. (Chew et al., 2011; Rodenko et al., 2006) A 384-well
clear flat bottom polystyrene microplate (Corning) was precoated
with 50 .mu.l of streptavidin (Invitrogen) at 2 .mu.g mL.sup.-1 in
PBS. Following 2 h of incubation at 37.degree. C., the wells were
washed with 0.05% Tween 20 in PBS (four times, 50 .mu.L) wash
buffer, treated with 50 .mu.l of blocking buffer (2% BSA in PBS),
and incubated 30 min at room temperature. Subsequently, 25 .mu.l of
peptide-exchanged samples that were 300.times. diluted with 20 mM
Tris HCl/50 mM NaCl were added in quadruplicate. The samples were
incubated for 15 min at RT, washed with 0.05% Tween wash buffer
(4.times.50 L), treated for 15 min with 25 .mu.L of HRP-conjugated
anti-02m (1 .mu.g mL.sup.-1 in PBS) at RT, washed with 0.05% Tween
wash buffer (4.times.50 .mu.L), and developed for 10-15 min with 25
L of ABTS-solution (Invitrogen), and the reactions were stopped by
the addition of 12.5 .mu.L of stop buffer (0.01% sodium azide in
0.1 M citric acid). Absorbance was subsequently measured at 415 nm
using a spectrophotometer (SpectraMax i3x; Molecular Devices).
[1011] Results for the G2 counterscreen "minipool" and G2 target
are shown in FIG. 65B. All three counterscreen peptides and the G2
peptide rescued the HLA complex from dissociation.
[1012] Results for the additional G2 "complete" pool counterscreen
peptides are shown in FIG. 66, demonstrating that they also form
stable HLA-peptide complexes.
[1013] Results for the G7 counterscreen "minipool" and G7 target
are shown in FIG. 69B. All three counterscreen peptides and the G7
peptide rescued the HLA complex from dissociation.
[1014] Results for the additional G7 "complete" pool counterscreen
peptides are shown in FIG. 68, demonstrating that they also form
stable HLA-peptide complexes.
[1015] Phage Library Screening
[1016] Phage library screening was carried out according to the
overall screening design described above. Three to four rounds of
bead-based panning were performed to identify scFv binders to each
peptide-HLA complex. For each round of panning, an aliquot of
starting phage was set aside for input titering and the remaining
phage was depleted three times against Dynabead M-280 streptavidin
beads (Life Technologies) followed by a depletion against
Streptavidin beads pre-bound with 100 pmoles of pooled negative
peptide-HLA complexes. For the first round of panning, 100 pmoles
of peptide-HLA complex bound to streptavidin beads was incubated
with depleted phage for 2 hours at room temperature with rotation.
Three five-minute washes with 0.5% BSA in 1.times.PBST (PBS+0.05%
Tween-20) followed by three five-minute washes with 0.5% BSA in
1.times.PBS were utilized to remove any unbound phage to the
peptide-HLA complex bound beads. To elute the bound phage from the
washed beads, 1 ml 0.1M TEA was added and incubated for 10 minutes
at room temperature with rotation. The eluted phage was collected
from the beads and neutralized with 0.5 ml 1M Tris-HCl pH 7.5. The
neutralized phage was then used to infect log growth TG-1 cells
(OD.sub.600=0.5) and after an hour of infection at 37.degree. C.,
cells were plated onto 2YT media with 100 .mu.g/ml carbenicillin
and 2% glucose (2YTCG) agar plates for output titer and bacterial
growth for subsequent panning rounds. For subsequent rounds of
panning, selection antigen concentrations were lowered while washes
increased by amount and length of wash times at show in Table
31.
TABLE-US-00052 TABLE 31 Phage library screening strategy Round
Antigen concentration Washes R1 100 pmol 3X PBST + 3X PBS (5 min
washes) R2 25 pmol 5 PBST (2x 30 sec, 3x 5 min) + 5 PBS (2x 30 sec,
3x 5 min) R3 10 pmol 8 PBST (4x 30 sec, 4x 5 min) + 8 PBS (4x 30
sec, 4x 5 min) R4 5 pmol, 10 pmol 30 min PBST + 30 min PBS
[1017] Individual scFvs were cloned from phage and sequenced by DNA
Sanger sequencing ("Sequence Unique Binders"). The individual scFvs
were also expressed in E. coli and periplasmic extracts (PPE) from
E. coli containing the individual crude scFvs were subjected to
scFv ELISA
[1018] scFv Periplasmic Extract (PPE) ELISA
[1019] The individual scFv cloned from phage obtained in the final
round of panning, and expressed in E. coli, was subjected to scFv
PPE ELISA as follows.
[1020] 96-well and/or 384-well streptavidin coated plates (Pierce)
were coated with 2 ug/ml peptide-HLA complex in HLA buffer and
incubated overnight at 4.degree. C. Plates were washed three times
between each step with PBST (PBS+0.05%). The antigen coated plates
were blocked with 3% BSA in PBS (blocking buffer) for 1 hour at
room temperature. After washing, scFv PPEs were added to the plates
and incubated at room temperature for 1 hour. Following washing,
mouse anti-v5 antibody (Invitrogen) in blocking buffer was added to
detect scFv and incubated at room temperature for 1 hour. After
washing, HRP-goat anti-mouse antibody (Jackson ImmunoResearch) was
added and incubated at room temperature for 1 hour. The plates were
then washed three times with PBST and 3 times with PBS before HRP
activity was detected with TMB 1-component Microwell Peroxidase
Substrate (Seracare) and neutralized with 2N sulfuric acid.
[1021] For negative peptide-HLA complex counter-screening, scFv PPE
ELISAs were performed as described above, except for the coating
antigen. HLA mini-pools consisted of 2 ug/ml of each of the three
negative peptide-HLA complexes pooled together and coated onto
streptavidin plates for comparison binding to their particular
peptide-HLA complex. HLA big pools consisted of 2 ug/ml of each of
all 18 negative peptide-HLA complexes pooled together and coated
onto streptavidin plates for comparison binding to their particular
peptide-HLA complex.
[1022] Those scFvs that showed selectivity for target pHLA compared
to negative control pHLA by scFv-ELISA as crude PPE, were
separately expressed and purified. The purified scFvs were titrated
by scFv ELISA for confirmation of binding only target pHLA compared
to negative control pHLA ("Selective Binders").
[1023] Clones were formatted into IgG, Fab, or scFv for further
biochemical and functional analysis. ScFv clones selected for Fab
production to be used for crystallization with their corresponding
pHLA complexes were selected based on several parameters: sequence
diversity, binding affinity, selectivity, and CDR3 diversity. The
clustal software was used to produce a dendrogram and assess the
sequence diversity of the Fab clones. The canonical 3D structures
of the scFv sequences, based on the VH type, were also considered
when possible. Binding affinity, as determined by the equilibrium
dissociation constant (KD), was measured using an Octet HTX
(ForteBio). Selectivity for the specific peptide-HLA complexes was
determined with an ELISA titration of the purified scFvs and
compared to negative peptides or streptavidin alone. Cutoff values
for the KD and selectivity were determined for each target set
based on the range of values obtained for the Fabs within each set.
Final clones were then selected to obtain the highest diversity in
sequence families and CDR3.
[1024] Table 32 shows the hit rate for the screening campaign
described above.
TABLE-US-00053 TABLE 32 hit rate for screening campaigns Group G2
G7 Gene target CT83 CT83 HLA A*01:01 A*02:01 Restricted peptide
NTDNNLAVY LLASSILCA # Sequence Unique 74 8 Binders # Selective
Binders 27 6 # selected for IgG 20 8 # selected for Fab 6 3 #
selected for scFv 20 7
[1025] Table 33 shows the VH and VL sequences of the G2 scFv
Selective Binders, selective for HLA-PEPTIDE Target
HLA-A*01:01_NTDNNLAVY
[1026] Table 34 shows the CDR sequences for the G2 Selective
Binders, selective for HLA-PEPTIDE Target HLA-A*01:01_NTDNNLAVY
CDRs were determined according to the Kabat numbering system._
[1027] Table 35 shows the VH and VL sequences of the G7 scFv
Selective Binders, selective for HLA-PEPTIDE Target
HLA-A*02:01_LLASSILCA.
[1028] Table 36 shows the CDR sequences for the G7 Selective
Binders, selective for HLA-PEPTIDE Target HLA-A*02:01_LLASSILCA.
CDRs were determined according to the Kabat numbering system.
Example 20: Isolation of TCRs that Specifically Bind HLA-PEPTIDE
Targets
[1029] FIG. 81 depicts an experimental workflow by which TCRs which
specifically bind HLA-PEPTIDE targets were isolated. Briefly, naive
CD8+ T cells that bind to the HLA-PEPTIDE target were isolated by
flow cytometry and polyclonally expanded. Following expansion,
specificity of cells for HLA-PEPTIDE target complex was tested by
flow cytometry. If a large fraction (>75%) of an expanded
population was specific for the HLA-PEPTIDE target, the population
as a whole was sequenced as a whole to identify TCRs.
Alternatively, cells that specifically bound the HLA-PEPTIDE target
were resorted, and only cells isolated after resort were sequenced.
TCR sequences were cloned into expression vectors and introduced
into recipient T cells as recombinant TCRs. Expression of the
evaluated TCR and binding of cognate HLA-PEPTIDE target complex by
the TCR-recombinant T cells was assessed.
[1030] Identified HLA-PEPTIDE Targets were Readily Recognized by
CD8+ T Cells
[1031] Peripheral Blood Mononuclear Cells (PBMCs) from healthy
donors were magnetically enriched for naive CD8+ T cells as
follows. PBMCs were obtained by processing leukapheresis samples
from healthy donors. Frozen PBMCs were thawed and incubated with
cocktail of biotinylated CD45RO, CD14, CD15, CD16, CD19, CD25,
CD34, CD36, CD57, CD123, anti-HLA-DR, CD235a (Glycophorin A),
CD244, and CD4 antibodies and were subsequently magnetically
labeled with anti-biotin microbeads for removal from PBMC
population. Enriched naive CD8 T cells were labelled with tetramers
comprising of target peptide and appropriate HLA molecule, stained
with live/dead and lineage markers and sorted by flow cytometry
according to the gating procedure depicted in FIG. 82. Cells that
bound the HLA-PEPTIDE tetramers were isolated. Following polyclonal
expansion, specificity of expanded CD8+ T cells was reassessed by
labeling with the HLA-PEPTIDE or no tetramer control. Flow
cytometry results for exemplary HLA-PEPTIDE targets
B*44:02_GEMSSNSTAL and A*01:01_EVDPIGHLY are shown in FIG. 83. Flow
cytometry results for the HLA-PETPIDE target A*03:01_GVHGGILNK is
shown in FIG. 84.
[1032] The number of isolated CD8+ T cells per HLA-PEPTIDE target
per donor and distribution of isolated CD8+ T cells frequency per
HLA-PEPTIDE target across all donors tested is shown in FIGS. 85A
(number of isolated CD8+ T cells) and 85B (frequency). Total number
of isolated naive CD8+ T cells per target ranged from 23-4181
antigen specific cells, which is in line with precursor frequencies
of T cells specific for known immunogenic viral antigens. These
cells present the source of natural TCRs for sequencing and further
characterization.
[1033] The number of isolated target-specific T cells per target
summarized across all tested donors is shown in Table 37
TABLE-US-00054 TABLE 37 number of isolated target-specific T cells
per target summarized across all donors Cumulative Number of TCR
Target Gene Source Cells Per Target EVDPIGHLY MAGEA3 5242
(HLA-A*0101) EVDPIGHVY MAGEA6 1296 (HLA-A*0101 GEMS SNSTAL CT83 48
(HLA-B*4402) GVHGGILNK PFN3 219 (HLA-A*0301) GVYDGEEHSV MAGEB2 17
(HLA-A*0201) LLASSILCA CT83 1665 (HLA-A*0201) LVIDTVTEV SPERT 16
(HLA-A*0201) NTDNNLAVY CT83 575 (HLA-A*0101)
[1034] These data demonstrate that identified HLA-PEPTIDE targets
are biologically relevant, as natural CD8+ T cells exist in HLA
matched human blood which bind/recognize target peptides in the
context of predicted associated MHC molecule.
[1035] CD8+ T Cells Yielded a Diverse Repertoire of Unique TCRs
which Bound the HLA-PEPTIDE Targets
[1036] Criteria for Sequencing of T-Cells
[1037] If a large fraction (>75%) of an expanded population was
specific for the HLA-PEPTIDE target, the population as a whole was
sequenced as a whole to identify TCRs. Then, selected TCR sequences
from the population were cloned into expression vectors and
transfected into recipient T-cells for confirmation of specificity.
Alternatively, cells that specifically bound the HLA-PEPTIDE target
were resorted, and only cells isolated after resort were
sequenced.
[1038] Sequencing Protocol
[1039] T cells isolated and expanded as described in FIG. 82 were
sequenced using 10.times. Genomics single cell resolution paired
immune TCR profiling approach. Specifically, two-to-eight thousand
live T cells were partitioned into single cell emulsions for
subsequent single cell cDNA generation and full-length TCR
profiling (5' UTR through constant region--ensuring alpha and beta
pairing). One approach utilizes a molecularly barcoded template
switching oligo at the 5' end of the transcript, a second approach
utilizes a molecularly barcoded constant region oligo at the 3'
end, and a third approach couples an RNA polymerase promoter to
either the 5' or 3' end of a TCR. All of these approaches enable
the identification and deconvolution of alpha and beta TCR pairs at
the single-cell level. The resulting barcoded cDNA transcripts
underwent an optimized enzymatic and library construction workflow
to reduce bias and ensure accurate representation of clonotypes
within the pool of cells. Libraries were sequenced on Illumina's
MiSeq or HiSeq4000 instruments (paired-end 150 cycles) for a target
sequencing depth of about five to fifty thousand reads per
cell.
[1040] Sequencing reads were processed through the 10.times.
provided software Cell Ranger. Sequencing reads were tagged with a
Chromium cellular barcodes and UMIs, which were used to assemble
the V(D)J transcripts cell by cell. The assembled contigs for each
cell were then annotated by mapping the assembled contigs to V(D)J
reference sequences from Ensembl version 87
(http://www.ensembl.org/).
[1041] Clonotypes were defined as alpha, beta chain pairs of unique
CDR3 amino acid sequences. Clonotypes were filtered for single
alpha and single beta chain pairs present at frequency above 2
cells to yield the final list of clonotypes per target peptide in a
specific donor. FIG. 86A depicts the number of unique TCR
clonotypes per HLA-PEPTIDE target for each tested donor. FIG. 86B
depicts the total number of unique clonotypes per HLA-PEPTIDE
target, summed across all donors tested.
[1042] TCR Sequences of Unique Clonotypes from Resorted Cells
[1043] Annotated variable, diversity, joining, and constant regions
of TCR clonotypes specific for A*0101_EVDPHIGHLY, from resorted
cells, are shown in Table 9 of PCT/US2018/046997, filed on Aug. 17,
2018, which application is incorporated by reference in its
entirety.
[1044] V(D)J and CDR3 sequences of .alpha. and .beta. chains of the
TCR clonotypes specific for A*0101_EVDPHIGHLY are shown in Table 10
of PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety.
[1045] Annotated variable, diversity, joining, and constant regions
of TCR clonotypes that demonstrated confirmed specificity in
recipient T-cells is shown in Table 11 of PCT/US2018/046997, filed
on Aug. 17, 2018, which application is incorporated by reference in
its entirety.
[1046] V(D)J and CDR3 sequences of .alpha. and .beta. chains of TCR
clonotypes that demonstrated confirmed specificity in recipient
T-cells is shown in Table 12 of PCT/US2018/046997, filed on Aug.
17, 2018, which application is incorporated by reference in its
entirety.
[1047] A table of the annotated reference .alpha. variable (TRAV),
.alpha. joining (TRAJ), .alpha. constant (TRAC), .beta. variable
(TRBV), .beta. diversity (TRBD), .beta. joining (TRBJ), and .beta.
constant (TRBC) sequences and their corresponding Ensembl
transcript (ENST) reference number is shown in Table 13 of
PCT/US2018/046997, filed on Aug. 17, 2018, which application is
incorporated by reference in its entirety. For any of the TCRs
disclosed, amino acid sequences that are at least 95%, at least
96%, at least 97%, and least 98%, at least 99%, or more than 99%
identical to the annotated reference sequences as disclosed
herein.
Example 21: T Cell Line Transiently Transfected with Identified
TCRs Specifically Bind to their Target HLA-PEPTIDE Complex, but not
to Negative Control Peptide-HLAs
[1048] Jurkat TIB-152 T cell line cultures were co-transfected with
a plasmid expressing human CD8 and a plasmid expressing TCR .alpha.
and .beta. chains with a GFP reporter gene using Nucleofector 4D
electroporator. Plasmids used for transfection are described in
FIGS. 49 and 50. 24-48 hours post transfection, Jurkat T cells were
analyzed for expression of the TCR of interest. Cells were assessed
for binding to HLA-PEPTIDE complexes and a control
infectious-disease-based peptide tetramer using flow cytometry.
Total population was gated on live single GFP-expressing cells
before evaluating binding of HLA-PEPTIDE target tetramer. FIG. 87
shows examples of Jurkat cells expressing A*0201_LLASSILCA-,
A*0201_GVYDGEEHSV-, B*4402_GEMSSNSTAL-, and
A*0101_EVDPIGHLY-specific TCRs binding to their respective
HLA-PEPTIDE targets but not to the control peptide tetramer.
Example 22: TCRs Cloned into a Viral Vector are Stably Expressed in
Primary Human CD8+ T Cells and Bind Cognate Peptide Target-MHC
Complexes
[1049] Lentiviral vectors were generated for TCR specific for the
HLA-PEPTIDE target HLA-A*0201_LLASSILCA. As a model vector system,
we used commercially available 3.sup.rd generation lentivirus base
expression vector system from System Biosciences, Palo Alto, Calif.
See FIG. 89.
[1050] Primary human CD8+ T cells were isolated and transduced with
the recombinant TCR lentivirus at multiplicity of infection
(MOI.about.10). T cells were expanded using rapid expansion
protocol for 1-2 weeks before assessment of TCR expression on CD8 T
cells by tetramer staining.
[1051] FIG. 88 depicts the gating strategy and flow data
demonstrating that transduced human CD8+ cells bind to the
HLA-PEPTIDE target.
Example 23: In Vivo Proof-of-Concept
[1052] Lead antibody or CAR-T constructs are evaluated in vivo to
demonstrate directed tumor killing in humanized mouse tumor models.
Lead antibody or CAR-T constructs are evaluated in xenograft tumor
models engrafted with human tumors and PBMCs. Anti-tumor activity
is measured and compared to control constructs to demonstrate
target-specific tumor killing.
[1053] While the invention has been particularly shown and
described with reference to a preferred embodiment and various
alternate embodiments, it will be understood by persons skilled in
the relevant art that various changes in form and details can be
made therein without departing from the spirit and scope of the
invention.
[1054] All references, issued patents and patent applications cited
within the body of the instant specification are hereby
incorporated by reference in their entirety, for all purposes.
Sequences
TABLE-US-00055 [1055] TABLE 4 VH and VL sequences of scFv hits that
bind target G5 Target Clone group name V.sub.H V.sub.L G5 G5_P7_
QVQLVQSGAEVKKPGASVKVSCK DIVMTQSPLSLPVTPGEPASISCRSS E7
ASGYTFTSYDINWVRQAPGQGLE QSLLHSNGYNYLDWYLQKPGQSP
WMGIINPRSGSTKYAQKFQGRVT QLLIYLGSYRASGVPDRFSGSGSGT
MTRDTSTSTVYMELSSLRSEDTAV DFTLKISRVEAEDVGVYYCMQGL
YYCARDGVRYYGMDVWGQGTTV QTPITFGQGTRLEIK TVSS G5 G5_P7_
QVQLVQSGAEVKKPGSSVKVSCK DIVMTQSPLSLPVTPGEPASISCRSS B3
ASGYTFTSHDINWVRQAPGQGLE QSLLHSNGYNYLDWYLQKPGQSP
WMGWMNPNSGDTGYAQKFQGR QLLIYLGSSRASGVPDRFSGSGSGT
VTITADESTSTAYMELSSLRSEDTA DFTLKISRVEAEDVGVYYCMQAL
VYYCARGVRGYDRSAGYWGQGT QTPPTFGPGTKVDIK LVIVSS G5 G5_P7_
EVQLLESGGGLVKPGGSLRLSCAA DIQMTQSPSSLSASVGDRVTITCQA A5
SGFSFSSYWMSWVRQAPGKGLEW SQDISNYLNWYQQKPGKAPKLLIY
ISYISGDSGYTNYADSVKGRFTISR AASSLQSGVPSRFSGSGSGTDFTLT
DDSKNTLYLQMNSLKTEDTAVYY ISSLQPEDFATYYCQQAISFPLTFG
CASHDYGDYGEYFQHWGQGTLV QSTKVEIK TVSS G5 G5_P7_
EVQLLQSGGGLVQPGGSLRLSCAA DIQMTQSPSSLSASVGDRVTITCRA F6
SGFTFSNSDMNWVRQAPGKGLEW SQSISSWLAWYQQKPGKAPKLLIY
VAYISSGSSTIYYADSVKGRFTISR SASTLQSGVPSRFSGSGSGTDFTLT
DNSKNTLYLQMNSLRAEDTAVYY ISSLQPEDFATYYCQQANSFPLTFG
CARVSWYCSSTSCGVNWFDPWGQ GGTKVEIK GTLVTVSS G5 G5-
EVQLLESGGGLVQPGGSLRLSCAA DIQMTQSPSSLSASVGDRVTITCRA P1B12
SGFTFSNSDMNWVRQAPGKGLEW SQSISSWLAWYQQKPGKAPKLLIY
VASISSSGGYINYADSVKGRFTISR AASSLQSGVPSRFSGSGSGTDFTLT
DNSKNTLYLQMNSLRAEDTAVYY ISSLQPEDFATYYCQQANSFPLTFG
CAKVNWNDGPYFDYWGQGTLVT GGTKVEIK VSS G5 G5- QVQLVQSGAEVKKPGSSVKVSCK
DIQMTQSPSSLSASVGDRVTITCRA P1C12 ASGGTFSNFGVSWLRQAPGQGLE
SQSISSWLAWYQQKPGKAPKLLIY WMGGIIPILGTANYAQKFQGRVTI
AASTLQSGVPSRFSGSGSGTDFTLT TADESTSTAYMELSSLRSEDTAVY
ISSLQPEDFATYYCQQSYSIPLTFG YCATPTNSGYYGPYYYYGMDVW GGTKVEIK
GQGTTVTVSS G5 G5-P1- QVQLVQSGAEVKKPGASVKVSCK
DIQMTQSPSSLSASVGDRVTITCRA E05 ASGYTFTSYNMHWVRQAPGQGLE
SQGISNYLNWYQQKPGKAPKLLIY WMGWINPNSGGTNYAQKFQGRV
YASSLQSGVPSRFSGSGSGTDFTLT TMTRDTSTSTVYMELSSLRSEDTA
ISSLQPEDFATYYCQQTYMMPYTF VYYCARDVMDVWGQGTTVTVSS GQGTKVEIK G5 G5-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P3G01
ASGGTFSGYLVSWVRQAPGQGLE SQSISSYLNWYQQKPGKAPKLLIY
WMGWINPNSGGTNTAQKFQGRVT GASSLQSGVPSRFSGSGSGTDFTLT
MTRDTSTSTVYMELSSLRSEDTAV ISSLQPEDFATYYCQQSYITPWTFG
YYCAREGYGMDVWGQGTTVTVS QGTKVEIK S G5 G5- QVQLVQSGAEVKKPGASVKVSCK
DIQMTQSPSSLSASVGDRVTITCRA P3G08 ASGYIFRNYPMHWVRQAPGQGLE
SQGISNYLAWYQQKPGKAPKLLIY WMGWINPDSGGTKYAQKFQGRV
AASSLQSGVPSRFSGSGSGTDFTLT TMTRDTSTSTVYMELSSLRSEDTA
ISSLQPEDFATYYCQQSYITPYTFG VYYCARDNGVGVDYWGQGTLVT QGTKLEIK VSS G5
G5- QVQLVQSGAEVKKPGASVKVSCK DIVMTQSPDSLAVSLGERATINCK P4B02
ASGYTFTGYYMHWVRQAPGQGL TSQSVLYRPNNENYLAWYQQKPG
EWMGWMNPNIGNTGYAQKFQGR QPPKLLIYQASIREPGVPDRFSGSG
VTMTRDTSTSTVYMELSSLRSEDT SGTDFTLTISSLQAEDVAVYYCQQ
AVYYCARGIADSGSYYGNGRDYY YYTTPYTFGQGTKLEIK YGMDVWGQGTTVTVSS G5 G5-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P4E04
ASGGTFSSYGISWVRQAPGQGLE SQSISRFLNWYQQKPGKAPKLLIY
WMGWINPNSGVTKYAQKFQGRV GASRPQSGVPSRFSGSGSGTDFTLT
TMTRDTSTSTVYMELSSLRSEDTA ISSLQPEDFATYYCQQSYSTPLTFG
VYYCARGDYYFDYWGQGTLVTV QGTKVEIK SS G5 G5R4- QVQLVQSGAEVKKPGASVKVSCK
DIVMTQSPLSLPVTPGEPASISCRSS P1D06 ASGYTFTSYDINWVRQAPGQGLE
QSLLHSNGYNYLDWYLQKPGQSP WMGWINPNSGDTKYSQKFQGRVT
QLLIYLGSHRASGVPDRFSGSGSGT MTRDTSTSTVYMELSSLRSEDTAV
DFTLKISRVEAEDVGVYYCMQAL YYCARDGTRYYGMDVWGQGTTV QTPLTFGGGTKVEIK TVSS
G5 G5R4- EVQLLESGGGLVKPGGSLRLSCAA EIVMTQSPATLSVSPGERATLSCRA P1H11
SGFTFSDYYMSWVRQAPGKGLEW SQSVSSNLAWYQQKPGQAPRLLIY
VSYISSSSSYTNYADSVKGRFTISR AASARASGIPARFSGSGSGTEFTLT
DDSKNTLYLQMNSLKTEDTAVYY ISSLQSEDFAVYYCQQYGSWPRTF
CARDVVANFDYWGQGTLVTVSS GQGTKVEIK G5 G5R4- QVQLVQSGAEVKKPGASVKVSCK
DIQMTQSPSSLSASVGDRVTITCRA P2B10 ASGGTFSSYAISWVRQAPGQGLE
SQSISSYLNWYQQKPGKAPKLLIY WMGWMNPDSGSTGYAQRFQGRV
GASRLQSGVPSRFSGSGSGTDFTLT TMTRDTSTSTVYMELSSLRSEDTA
ISSLQPEDFATYYCQQSYSTPVTFG VYYCARGHSSGWYYYYGMDVW QGTKVEIK GQGTTVTVSS
G5 G5R4- EVQLLESGGGLVQPGGSLRLSCAA DIVMTQSPLSLPVTPGEPASISCRSS P2H8
SGFTFTSYSMHWVRQAPGKGLEW QSLLHSNGYNYLDWYLQKPGQSP
VSSITSFTNTMYYADSVKGRFTISR QLLIYLGSNRASGVPDRFSGSGSGT
DNSKNTLYLQMNSLRAEDTAVYY DFTLKISRVEAEDVGVYYCMQAL
CAKDLGSYGGYYWGQGTLVTVSS QTPYTFGQGTKVEIK G5 G5R4-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCQA P3G05
ASGYTFTNYYMHWVRQAPGQGL SEDISNHLNWYQQKPGKAPKLLIY
EWMGIINPSGGSTSYAQKFQGRVT DALSLQSGVPSRFSGSGSGTDFTLT
MTRDTSTSTVYMELSSLRSEDTAV ISSLQPEDFATYYCQQANSFPFTFG
YYCARSWFGGFNYHYYGMDVWG PGTKVDIK QGTTVTVSS G5 G5R4-
QVQLVQSGAEVKKPGASVKVSCK DIVMTQSPLSLPVTPGEPASISCRSS P4A07
ASGYTFTSYYMHWVRQAPGQGLE QSLLHSNGYNYLDWYLQKPGQSP
WMGWMNPNSGNTGYAQKFQGR QLLIYLGSNRASGVPDRFSGSGSGT
VTMTRDTSTSTVYMELSSLRSEDT DFTLKISRVEAEDVGVYYCMQAL
AVYYCARELPIGYGMDVWGQGTT QTPLTFGQGTKVEIK VTVSS G5 G5R4-
QVQLVQSGAEVKKPGSSVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P4B01
ASGGTFSSYAISWVRQAPGQGLE SQSISSYLNWYQQKPGKAPKLLIY
WMGGIIPIVGTANYAQKFQGRVTI AASSLQSGVPSRFSGSGSGTDFTLT
TADESTSTAYMELSSLRSEDTAVY ISSLQPEDFATYYCQQSYSTPLTFG
YCARGGSYYYYGMDVWGQGTTV GGTKVEIK TVSS
TABLE-US-00056 TABLE 5 CDR sequences of identified scFvs to G5,
numbered according to the Kabat numbering scheme Target Clone group
name HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 G5 G5_P7_ YTFTS GIINPRS
CARDGVR RSSQSLLH LGSYR CMQGLQ E7 YDIN GSTKYA YYGMDV SNGYNYL AS
TPITF W D G5 G5_P7_ YTFTS GWMNP CARGVRG RSSQSLLH LGSSR CMQALQ B3
HDIN NSGDTG YDRSAGY SNGYNYL AS TPPTF YA W D G5 G5_P7_ FSFSSY
SYISGDS CASHDYG QASQDISN AASSL CQQAISF A5 WMS GYTNYA DYGEYFQ YLN QS
PLTF HW G5 G5_P7_ FTFSNS AYISSGS CARVSWY RASQSISS SASTLQ CQQANS F6
DMN STIYYA CSSTSCGV WLA S FPLTF NWFDPW G5 G5- FTFSNS ASISSSG CAKVNW
RASQSISS AASSL CQQANS P1B12 DMN GYINYA NDGPYFD WLA QS FPLTF YW G5
G5- GTFSNF GGIIPILG CATPTNS RASQSISS AASTL CQQSYSI P1C12 GVS TANYA
GYYGPYY WLA QS PLTF YYGMDV W G5 G5-P1- YTFTS GWINPN CARDVM RASQGISN
YASSL CQQTYM E05 YNMH SGGTNY DVW YLN QS MPYTF A G5 G5- GTFSG GWINPN
CAREGYG RASQSISS GASSL CQQSYIT P3G01 YLVS SGGTNT MDVW YLN QS PWTF A
G5 G5- YIFRNY GWINPD CARDNGV RASQGISN AASSL CQQSYIT P3G08 PMH
SGGTKY GVDYW YLA QS PYTF A G5 G5- YTFTG GWMNP CARGIAD KTSQSVL
QASIRE CQQYYT P4B02 YYMH NIGNTG SGSYYGN YRPNNEN P TPYTF YA GRDYYYG
YLA MDVW G5 G5- GTFSSY GWINPN CARGDYY RASQSISR GASRP CQQSYS P4E04
GIS SGVTKY FDYW FLN QS TPLTF A G5 G5R4- YTFTS GWINPN CARDGTR
RSSQSLLH LGSHR CMQALQ P1D06 YDIN SGDTKY YYGMDV SNGYNYL AS TPLTF S W
D G5 G5R4- FTFSDY SYISSSSS CARDVVA RASQSVSS AASAR CQQYGS P1H11 YMS
YTNYA NFDYW NLA AS WPRTF G5 G5R4- GTFSSY GWMNP CARGHSS RASQSISS
GASRL CQQSYS P2B10 AIS DSGSTG GWYYYY YLN QS TPVTF YA GMDVW G5 G5R4-
FTFTSY SSITSFTN CAKDLGS RSSQSLLH LGSNR CMQALQ P2H8 SMH TMYYA YGGYYW
SNGYNYL AS TPYTF D G5 G5R4- YTFTN GIINPSG CARSWFG QASEDISN DALSL
CQQANS P3G05 YYMH GSTSYA GFNYHYY HLN QS FPFTF GMDVW G5 G5R4- YTFTS
GWMNP CARELPIG RSSQSLLH LGSNR CMQALQ P4A07 YYMH NSGNTG YGMDVW
SNGYNYL AS TPLTF YA D G5 G5R4- GTFSSY GGIIPVM CARGGSY RASQSISS
AASSL CQQSYS P4B01 AIS GTGNYA YYYGMD YLN QS TPLTF VW
TABLE-US-00057 TABLE 6 VH and VL sequences of scFv hits that bind
target G8 Target Clone group name V.sup.H V.sup.L G8 G8-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P1A03
ASGGTFSRSAITWVRQAPGQGLE SQSITSYLNWYQQKPGKAPKLLIY
WMGWINPNSGATNYAQKFQGRV DASNLETGVPSRFSGSGSGTDFTLT
TMTRDTSTSTVYMELSSLRSEDTA ISSLQPEDFATYYCQQNYNSVTFG
VYYCARDDYGDYVAYFQHWGQG QGTKLEIK TLVTVSS G8 G8-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCW P1A04
ASGYPFIGQYLHWVRQAPGQGLE ASQGISSYLAWYQQKPGKAPKLLI
WMGIINPSGDSATYAQKFQGRVT YAASSLQSGVPSRFSGSGSGTDFTL
MTRDTSTSTVYMELSSLRSEDTAV TISSLQPEDFATYYCQQSYNTPWT
YYCARDLSYYYGMDVWGQGTTV FGPGTKVDIK TVSS G8 G8-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P1A06
ASGYTFTNYYMHWVRQAPGQGL SQAISNSLAWYQQKPGKAPKLLIY
EWMGWMNPIGGGTGYAQKFQGR AASTLQSGVPSRFSGSGSGTDFTLT
VTMTRDTSTSTVYMELSSLRSEDT ISSLQPEDFATYYCGQSYSTPPTFG
AVYYCARVYDFWSVLSGFDIWGQ QGTKLEIK GTLVTVSS G8 G8-
EVQLLESGGGLVQPGGSLRLSCAA DIQMTQSPSSLSASVGDRVTITCRA P1B03
SGFTFSDYYMSWVRQAPGKGLEW SQSISSYLNWYQQKPGKAPKLLIY
VSGINWNGGSTGYADSVKGRFTIS KASSLESGVPSRFSGSGSGTDFTLT
RDNSKNTLYLQMNSLRAEDTAVY ISSLQPEDFATYYCQQSYSAPYTFG
YCARVEQGYDIYYYYYMDVWGK PGTKVDIK GTTVTVSS G8 G8-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCQA P1C11
ASGGTLSSYPINWVRQAPGQGLE SQDISNYLNWYQQKPGKAPKLLIY
WMGWISTYSGHADYAQKLQGRV AASSLQSGVPSRFSGSGSGTDFTLT
TMTRDTSTSTVYMELSSLRSEDTA ISSLQPEDFATYYCQQSYSIPPTFG
VYYCARSYDYGDYLNFDYWGQG GGTKVDIK TLVTVSS G8 G8-
EVQLLESGGGLVQPGGSLRLSCAA DIQMTQSPSSLSASVGDRVTITCQA P1D02
SGFTFSSYWMSWVRQAPGKGLEW SQDISNYLNWYQQKPGKAPKLLIY
VSSISGRGDNTYYADSVKGRFTISR AASSLQSGVPSRFSGSGSGTDFTLT
DNSKNTLYLQMNSLRAEDTAVYY ISSLQPEDFATYYCQQSYSAPYTFG
CARASGSGYYYYYGMDVWGQGT GGTKVEIK TVTVSS G8 G8-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P1H08
ASGYTFGNYFMHWVRQAPGQGLE SQGINSYLAWYQQKPGKAPKLLIY
WMGMVNPSGGSETFAQKFQGRVT DASNLETGVPSRFSGSGSGTDFTLT
MTRDTSTSTVYMELSSLRSEDTAV ISSLQPEDFATYYCQQHNSYPPTFG
YYCAASTWIQPFDYWGQGTLVTV QGTKLEIK SS G8 G8- EVQLLESGGGLVQPGGSLRLSCAA
DIQMTQSPSSLSASVGDRVTITCRA P2B05 SGFDFSIYSMNWVRQAPGKGLEW
SQSISRWLAWYQQKPGKAPKLLIY VSAISGSGGSTYYADSVKGRFTISR
AASSLQSGVPSRFSGSGSGTDFTLT DNSKNTLYLQMNSLRAEDTAVYY
ISSLQPEDFATYYCQQYSTYPITIG CASNGNYYGSGSYYNYWGQGTL QGTKVEIK VTVSS G8
G8- QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P2E06
ASGYTLTTYYMHWVRQAPGQGLE SQGISNSLAWYQQKPGKAPKLLIY
WMGWINPNSGGTNYAQKFQGRV AASSLQSGVPSRFSGSGSGTDFTLT
TMTRDTSTSTVYMELSSLRSEDTA ISSLQPEDFATYYCQQANSFPWTF
VYYCARAVYYDFWSGPFDYWGQ GQGTKLEIK GTLVTVSS G8 R3G8-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P2C10
ASGYTFTSYYMHWVRQAPGQGLE SQDVSTWLAWYQQKPGKAPKLLI
WMGWINPYSGGTNYAQKFQGRV YAASSLQSGVPSRFSGSGSGTDFTL
TMTRDTSTSTVYMELSSLRSEDTA TISSLQPEDFATYYCQQSHSTPQTF
VYYCAKGGIYYGSGSYPSWGQGT GQGTKVEIK LVTVSS G8 R3G8-
QVQLVQSGAEVKKPGSSVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P2E04
ASGGTFSSYGVSWVRQAPGQGLE SQSISSWLAWYQQKPGKAPKLLIY
WMGWISPYSGNTDYAQKFQGRVT DASNLETGVPSRFSGSGSGTDFTLT
ITADESTSTAYMELSSLRSEDTAVY ISSLQPEDFATYYCQQSYSTPLTFG
YCARGLYYMDVWGKGTTVTVSS GGTKLEIK G8 R3G8- QVQLVQSGAEVKKPGASVKVSCK
DIQMTQSPSSLSASVGDRVTITCRA P4F05 ASGYTFSNMYLHWVRQAPGQGLE
SQGISNYLAWYQQKPGKAPKLLIY WMGWINPNTGDTNYAQTFQGRV
AASTLQSGVPSRFSGSGSGTDFTLT TMTRDTSTSTVYMELSSLRSEDTA
ISSLQPEDFATYYCQQSYSTPLTFG VYYCARGLYGDYFLYYGMDVWG GGTKVEIK QGTKVTVSS
G8 R3G8- QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P5C03
ASGYTFTSYYMHWVRQAPGQGLE SQGISNWLAWYQQKPGKAPKLLI
WMGWMNPNSGNTGYAQKFQGR YAASTLQSGVPSRFSGSGSGTDFTL
VTMTRDTSTSTVYMELSSLRSEDT TISSLQPEDFATYYCQQTYSTPWTF
AVYYCARGLLGFGEFLTYGMDV GQGTKLEIK WGQGTLVTVSS G8 R3G8-
QVQLVQSGAEVKKPGASVKVSCK EIVMTQSPATLSVSPGERATLSCRA P5F02
ASGYTFTGYYIHWVRQAPGQGLE SQSVGNSLAWYQQKPGQAPRLLIY
WMGVINPSGGSTTYAQKLQGRVT GASTRATGIPARFSGSGSGTEFTLTI
MTRDTSTSTVYMELSSLRSEDTAV SSLQSEDFAVYYCQQYGSSPYTFG
YYCARDRDSSWTYYYYGMDVWG QGTKVEIK QGTTVTVSS G8 R3G8-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P5G08
ASGYTFTSNYMHWVRQAPGQGLE SQSISGYLNWYQQKPGKAPKLLIY
WMGWMNPNSGNTGYAQKFQGR AASSLQSGVPSRFSGSGSGTDFTLT
VTMTRDTSTSTVYMELSSLRSEDT ISSLQPEDFATYYCQQSHSTPLTFG
AVYYCARGLYGDYFLYYGMDVW QGTKVEIK GQGTTVTVSS G8 G8-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P1C01
ASGGTFSSHAISWVRQAPGQGLE SQNIYTYLNWYQQKPGKAPKLLIY
WMGVIIPSGGTSYTQKFQGRVTMT DASNLETGVPSRFSGSGSGTDFTLT
RDTSTSTVYMELSSLRSEDTAVYY ISSLQPEDFATYYCQQANGFPLTFG
CARGDYYDSSGYYFPVYFDYWGQ GGTKVEIK GTLVTVSS G8 G8-
QVQLVQSGAEVKKPGASVKVSCK DIQMTQSPSSLSASVGDRVTITCRA P2C11
ASGYTFTSYAMNWVRQAPGQGLE SQSISSYLNWYQQKPGKAPKLLIY
WMGWINPNSGGTNYAQKFQGRV AASSLQSGVPSRFSGSGSGTDFTLT
TMTRDTSTSTVYMELSSLRSEDTA ISSLQPEDFATYYCQQSYSTPLTFG
VYYCARDPFWSGHYYYYGMDVW GGTKVEIK GQGTTVTVSS
TABLE-US-00058 TABLE 7 CDR sequences of identified scFvs to G8,
numbered according to the Kabat numbering scheme Target Clone group
name HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 G8 G8- GTFSRS GWINPN
CARDDYG RASQSITS DASNL CQQNYN P1A03 AIT SGATNY DYVAYFQ YLN ET SVTF
A HW G8 G8- YPFIGQ GIINPSG CARDLSY WASQGISS AASSL CQQSYN P1A04 YLH
DSATYA YYGMDV YLA QS TPWTF W G8 G8- YTFTN GWMNPI CARVYDF RASQAISN
AASTL CGQSYS P1A06 YYMH GGGTGY WSVLSGF SLA QS TPPTF A DIW G8 G8-
FTFSDY SGINWN CARVEQG RASQSISS KASSLE CQQSYS P1B03 YMS GGSTGY
YDIYYYY YLN S APYTF A YMDVW G8 G8- GTLSS GWISTYS CARSYDY QASQDISN
AASSL CQQSYSI P1C11 YPIN GHADYA GDYLNFD YLN QS PPTF YW G8 G8-
FTFSSY SSISGRG CARASGS QASQDISN AASSL CQQSYS P1D02 WMS DNTYYA
GYYYYYG YLN QS APYTF MDVW G8 G8- YTFGN GMVNPS CAASTWI RASQGINS
DASNL CQQHNS P1H08 YFMH GGSETFA QPFDYW YLA ET YPPTF G8 G8- FDFSIY
SAISGSG CASNGNY RASQSISR AASSL CQQYST P2B05 SMN GSTYYA YGSGSYY WLA
QS YPITI NYW G8 G8- YTLTT GWINPN CARAVYY RASQGISN AASSL CQQANS
P2E06 YYMH SGGTNY DFWSGPF SLA QS FPWTF A DYW G8 R3G8- YTFTS GWINPY
CAKGGIY RASQDVS AASSL CQQSHS P2C10 YYMH SGGTNY YGSGSYP TWLA QS
TPQTF A SW G8 R3G8- GTFSSY GWISPYS CARGLYY RASQSISS DASNL CQQSYS
P2E04 GVS GNTDYA MDVW WLA ET TPLTF G8 R3G8- YTFSN GWINPN CARGLYG
RASQGISN AASTL CQQSYS P4F05 MYLH TGDTNY DYFLYYG YLA QS TPLTF A MDVW
G8 R3G8- YTFTS GWMNP CARGLLG RASQGISN AASTL CQQTYS P5C03 YYMH
NSGNTG FGEFLTY WLA QS TPWTF YA GMDVW G8 R3G8- YTFTG GVINPSG CARDRDS
RASQSVG GASTR CQQYGS P5F02 YYIH GSTTYA SWTYYYY NSLA AT SPYTF GMDVW
G8 R3G8- YTFTS GWMNP CARGLYG RASQSISG AASSL CQQSHS P5G08 NYMH
NSGNTG DYFLYYG YLN QS TPLTF YA MDVW G8 G8- GTFSSH GVIIPSG CARGDYY
RASQNIYT DASNL CQQANG P1C01 AIS GTSYT DSSGYYF YLN ET FPLTF PVYFDYW
G8 G8- YTFTS GWINPN CAKDPFW RASQSISS AASSL CQQSYS P2C11 YAMN SGGTNY
SGHYYYY YLN QS TPLTF A GMDVW
TABLE-US-00059 TABLE 8 VH and VL sequences of scFv hits that bind
target G10 Target Clone group name V.sub.H V.sub.L G10 R3G10-
EVQLLESGGGLVKPGGSLRLSCAAS DIQMTQSPSSLSASVGDRVTITCRAS P1A07
GFTFSSYWMSWVRQAPGKGLEWVS QGISNYLAWYQQKPGKAPKLLIYAAS
GISARSGRTYYADSVKGRFTISRDDS SLQGGVPSRFSGSGSGTDFTLTISSL
KNTLYLQMNSLKTEDTAVYYCARDQ QPEDFATYYCQQYFTTPYTFGQGTKL
DTIFGVVITWFDPWGQGTLVTVSS EIK G10 R3G10- QVQLVQSGAEVKKPGASVKVSCKAS
DIQMTQSPSSLSASVGDRVTITCRAS P1B07 GYTFTSYYMHWVRQAPGQGLEWMG
QSISRWLAWYQQKPGKAPKLLIFDAS IIHPGGGTTSYAQKFQGRVTMTRDTS
RLQSGVPSRFSGSGSGTDFTLTISSL TSTVYMELSSLRSEDTAVYYCARDKV
QPEDFATYYCQQAEAFPYTFGQGTK YGDGFDPWGQGTLVTVSS VEIK G10 R3G10-
QVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRAS P1E12
GYIFTGYYMHWVRQAPGQGLEWMG QSISSYLNWYQQKPGKAPKLLIYAAS
MIGPSDGSTSYAQKFQGRVTMTRDT SLQSGVPSRFSGSGSGTDFTLTISSL
STSTVYMELSSLRSEDTAVYYCARED QPEDFATYYCQQSYSTPITFGQGTRL
DSMDVWGKGTTVTVSS EIK G10 R3G10- QVQLVQSGAEVKKPGASVKVSCKAS
DIQMTQSPSSLSASVGDRVTITCRAS P1F06 GYTFIGYYMHWVRQAPGQGLEWMG
QSISNYLNWYQQKPGKAPKLLIYKAS MIGPSDGSTSYAQKFQGRVTMTRDT
SLESGVPSRFSGSGSGTDFTLTISSL STSTVYMELSSLRSEDTAVYYCARDS
QPEDFATYYCQQSYIIPYTFGQGTKL SGLDPWGQGTLVTVSS EIK G10 R3G10-
QVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRAS P1H01
GYTFTGYYMHWVRQAPGQGLEWMG QSISNYLNWYQQKPGKAPKLLIYAAS
MIGPSDGSTSYAQKFQGRVTMTRDT SLQSGVPSRFSGSGSGTDFTLTISSL
STSTVYMELSSLRSEDTAVYYCARGV QPEDFATYYCHQTYSTPLTFGQGTKV
GNLDYWGQGTLVTVSS EIK G10 R3G10- QVQLVQSGAEVKKPGASVKVSCKAS
DIQMTQSPSSLSASVGDRVTITCRAS P1H08 GVTFSTSAISWVRQAPGQGLEWMG
QGISNYLAWYQQKPGKAPKWYSAS WISPYNGNTDYAQMLQGRVTMTRDT
NLQSGVPSRFSGSGSGTDFTLTISSL STSTVYMELSSLRSEDTAVYYCARDA
QPEDFATYYCQQAYSFPVVTFGQGTK HQYYDFWSGYYSGTYYYGMDVWGQ VEIK GTTVTVSS
G10 R3G10- QVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRAS
P2C04 GGTFSNSIINWVRQAPGQGLEWMG QNISSYLNWYQQKPGKAPKLLIYAAS
WMNPNSGNTNYAQKFQGRVTMTRD SLQSGVPSRFSGSGSGTDFTLTISSL
TSTSTVYMELSSLRSEDTAVYYCARE QPEDFATYYCQQGYSTPLTFGQGTR
QWPSYWYFDLWGRGTLVTVSS LEIK G10 R3G10- QVQLVQSGAEVKKPGASVKVSCKAS
DIQMTQSPSSLSASVGDRVTITCRAS P2G11 GGTFSTHDINWVRQAPGQGLEWMG
QDISRYLAWYQQKPGKAPKLLIYDAS VINPSGGSAIYAQKFQGRVTMTRDTS
NLETGVPSRFSGSGSGTDFTLTISSL TSTVYMELSSLRSEDTAVYYCARDRG
QPEDFATYYCQQANSFPRTFGQGTK YSYGYFDYWGQGTLVTVSS VEIK G10 R3G10-
QVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCQAS P3E04
GNTFIGYYVHWVRQAPGQGLEWVGII QDISNYLNWYQQKPGKAPKLLIYAAS
NPNGGSISYAQKFQGRVTMTRDTST NLQSGVPSRFSGSGSGTDFTLTISSL
STVYMELSSLRSEDTAVYYCARGSG QPEDFATYYCQQANSLPYTFGQGTK
DPNYYYYYGLDVWGQGTTVTVSS VEIK G10 R3G10- QVQLVQSGAEVKKPGASVKVSCKAS
DIQMTQSPSSLSASVGDRVTITCRAS P4A02 GYTLSYYYMHWVRQAPGQGLEWMG
QSISSYLNWYQQKPGKAPKLLIYAAS MIGPSDGSTSYAQRFQGRVTMTRDT
TLQNGVPSRFSGSGSGTDFTLTISSL STGTVYMELSSLRSEDTAVYYCARDT
QPEDFATYYCQQSYSTPFTFGPGTK GDHFDYWGQGTLVTVSS VDIK G10 R3G10-
QVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRAS P4C05
GYTFTGYYMHWVRQAPGQGLEWMG QRISSYLNWYQQKPGKAPKWYSAS
IIGPSDGSTTYAQKFQGRVTMTRDTS TLQSGVPSRFSGSGSGTDFTLTISSL
TSTVYMELSSLRSEDTAVYYCARAEN QPEDFATYYCQQSYSTPFTFGPGTK
GMDVWGQGTTVTVSS VDIK G10 R3G10- QVQLVQSGAEVKKPGASVKVSCKAS
DIQMTQSPSSLSASVGDRVTITCRAS P4D04 GYTFTGYYVHWVRQAPGQGLEWMG
QSISSYLAWYQQKPGKAPKLLIYDAS IIAPSDGSTNYAQKFQGRVTMTRDTS
KLETGVPSRFSGSGSGTDFTLTISSL TSTVYMELSSLRSEDTAVYYCARDPG
QPEDFATYYCQQSYGVPTFGQGTKL GYMDVWGKGTTVTVSS EIK G10 R3G10-
QVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRAS P4D10
GYTFTGYYLHWVRQAPGQGLEWMG QGISSWLAWYQQKPGKAPKLLIYDAS
MIGPSDGSTSYAQKFQGRVTMTRDT NLETGVPSRFSGSGSGTDFTLTISSL
STSTVYMELSSLRSEDTAVYYCARDG QPEDFATYYCQQSYSTPLTFGGGTK
DAFDIWGQGTMVTVSS VEIK G10 R3G10- QVQLVQSGAEVKKPGSSVKVSCKAS
DIQMTQSPSSLSASVGDRVTITCRAS P4E07 GYTFTGYYMHWVRQAPGQGLEWMG
QSISSYLNWYQQKPGKAPKLLIYAAS RISPSDGSTTYAPKFQGRVTITADEST
SLQSGVPSRFSGSGSGTDFTLTISSL STAYMELSSLRSEDTAVYYCARDMG
QPEDFATYYCQQSYSTPLTFGGGTK DAFDIWGQGTTVTVSS VEIK G10 R3G10-
QVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRAS P4E12
GYTFTGYYMHWVRQAPGQGLEWMG QGISTYLAWYQQKPGKAPKLLIYDAS
MIGPSDGSTSYAQRFQGRVTMTRDT SLQSGVPSRFSGSGSGTDFTLTISSL
STSTVYMELSSLRSEDTAVYYCAREE QPEDFATYYCQQYYSYPVVTFGQGTR
DGMDVWGQGTTVTVSS LEIK G10 R3G10- QVQLVQSGAEVKKPGASVKVSCKAS
DIQMTQSPSSLSASVGDRVTITCRAS P4G06 GYTLSYYYMHWVRQAPGQGLEWMG
QSISSYLNWYQQKPGKAPKLLIYAAS MIGPSDGSTSYAQRFQGRVTMTRDT
TLQNGVPSRFSGSGSGTDFTLTISSL STGTVYMELSSLRSEDTAVYYCARDT
QPEDFATYYCQQSYSTPFTFGPGTK GDHFDYWGQGTLVTVSS VDIK G10 R3G10-
QVQLVQSGAEVKKPGSSVKVSCKAS DIVMTQSPLSLPVTPGEPASISCRSSQ P5A08
GGTFNNFAISWVRQAPGQGLEWMG SLLHSNGYNYLDWYLQKPGQSPQLLI
GIIPIFDATNYAQKFQGRVTFTADEST YLGSNRASGVPDRFSGSGSGTDFTL
STAYMELSSLRSEDTAVYYCARGEYS KISRVEAEDVGVYYCMQTLKTPLSFG
SGFFFVGWFDLWGRGTQVTVSS GGTKVEIK G10 R3G10-
QVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRAS P5C08
GYNFTGYYMHWVRQAPGQGLEWM QSISSYLNWYQQKPGKAPKLLIYAAS
GIIAPSDGSTNYAQKFQGRVTMTRDT SLQSGVPSRFSGSGSGTDFTLTISSL
STSTVYMELSSLRSEDTAVYYCARET QPEDFATYYCQQSYSTPLTFGGGTK
GDDAFDIWGQGTMVTVSS VEIK
TABLE-US-00060 TABLE 9 CDR sequences of identified scFvs to G10,
numbered according to the Kabat numbering scheme Target Clone group
name HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 G10 R3G10- FTFSSYW SGISARS
CARDQDTI RASQGISN AASSLQ CQQYFTT P1A07 MS GRTYYA FGVVITWF YLA G
PYTF DPW G10 R3G10- YTFTSYY GIIHPGG CARDKVYG RASQSISR DASRLQ
CQQAEAF P1B07 MH GTTSYA DGFDPW WLA S PYTF G10 R3G10- YIFTGYYM
GMIGPSD CAREDDS RASQSISS AASSLQ CQQSYST P1E12 H GSTSYA MDVW YLN S
PITF G10 R3G10- YTFIGYYM GMIGPSD CARDSSGL RASQSISN KASSLE CQQSYIIP
P1F06 H GSTSYA DPW YLN S YTF G10 R3G10- YTFTGYY GMIGPSD CARGVGNL
RASQSISN AASSLQ CHQTYST P1H01 MH GSTSYA DYW YLN S PLTF G10 R3G10-
VTFSTSAI GWISPYN CARDAHQ RASQGISN SASNLQ CQQAYSF P1H08 S GNTDYA
YYDFWSG YLA S PWTF YYSGTYYY GMDVW G10 R3G10- GTFSNSII GWMNPN
CAREQWP RASQNISS AASSLQ CQQGYS P2C04 N SGNTNYA SYWYFDL YLN S TPLTF
W G10 R3G10- GTFSTHDI GVINPSG CARDRGY RASQDISR DASNLE CQQANS P2G11
N GSAIYA SYGYFDY YLA T FPRTF W G10 R3G10- NTFIGYYV GIINPNG CARGSGD
QASQDISN AASNLQ CQQANSL P3E04 H GSISYA PNYYYYYG YLN S PYTF LDVW G10
R3G10- YTLSYYY GMIGPSD CARDTGD RASQSISS AASTLQ CQQSYST P4A02 MH
GSTSYA HFDYW YLN N PFTF G10 R3G10- YTFTGYY GIIGPSDG CARAENG
RASQRISS SASTLQ CQQSYST P4C05 MH STTYA MDVW YLN S PFTF G10 R3G10-
YTFTGYY GIIAPSDG CARDPGG RASQSISS DASKLE CQQSYG P4D04 VH STNYA
YMDVW YLA T VPTF G10 R3G10- YTFTGYYL GMIGPSD CARDGDAF RASQGISS
DASNLE CQQSYST P4D10 H GSTSYA DIW WLA T PLTF G10 R3G10- YTFTGYY
GRISPSD CARDMGD RASQSISS AASSLQ CQQSYST P4E07 MH GSTTYA AFDIW YLN S
PLTF G10 R3G10- YTFTGYY GMIGPSD CAREEDG RASQGIST DASSLQ CQQYYS
P4E12 MH GSTSYA MDVW YLA S YPVVTF G10 R3G10- YTLSYYY GMIGPSD
CARDTGD RASQSISS AASTLQ CQQSYST P4G06 MH GSTSYA HFDYW YLN N PFTF
G10 R3G10- GTFNNFAI GGIIPIFD CARGEYSS RSSQSLLH LGSNRA CMQTLKT P5A08
S ATNYA GFFFVGWF SNGYNYLD S PLSF DLW G10 R3G10- YNFTGYY GIIAPSDG
CARETGDD RASQSISS AASSLQ CQQSYST P5C08 MH STNYA AFDIW YLN S
PLTF
[1056] Table 15 (CDR3 Sequences for G10 TCRs)
[1057] This table is included in PCT/US2018/06793, filed on Dec.
28, 2018, which is incorporated by reference in its entirety.
[1058] Table 16: Full Length Alpha and Beta TCR Sequences (G10)
[1059] This table is included in PCT/US2018/06793, filed on Dec.
28, 2018, which is incorporated by reference in its entirety.
[1060] Table 18: CDR3 Sequences for TCR Clonotypes Specific for
HLA-PEPTIDE A*01:01 HSEVGLPVY
[1061] This table is included in PCT/US2018/06793, filed on Dec.
28, 2018, which is incorporated by reference in its entirety.
[1062] Table 19: Full Length Alpha V(J) and Beta V(D)J Sequences of
Identified TCR Clonotypes Specific for HLA-PEPTIDE A*01:01
HSEVGLPVY
[1063] This table is included in PCT/US2018/06793, filed on Dec.
28, 2018, which is incorporated by reference in its entirety.
TABLE-US-00061 TABLE 33 VH and VL sequences for G2 scFv Selective
Binders, selective for HLA-PEPTIDE Target HLA-A*01:01 NTDNNLAVY.
Target Clone group name V.sub.H V.sub.L G2 G2- QVQLVQSGAEVKKPGASVKV
DIQMTQSPSSLSASVGDRVTI P2E07 SCKASGGTFSSATISWVRQAP
TCRASQSISTWLAWYQQKPG GQGLEWMGWIYPNSGGTVY KAPKLLIYAASSLRSGVPSRF
AQKFQGRVTMTRDTSTSTVY SGSGSGTDFTLTISSLQPEDFA MELSSLRSEDTAVYYCAATE
TYYCQQSYNTPYTFGQGTKL WLGVWGQGTTVTVSS EIK G2 G2-
EVQLLQSGAEVKKPGSSVKV DIQMTQSPSSLSASVGDRVTI P2E03
SCKASGGTFSSYAISWVRQAP TCRASQSISRWLAWYQQKPG GQGLEWMGWINPNSGGTISA
KAPKLLIYAASTVQSGVPSRF PNFQGRVTMTRDTSTSTVYM SGSGSGTDFTLTISSLQPEDFA
ELSSLRSEDTAVYYCARANW TYYCQQSYSTPYTFGQGTKL LDYWGQGTLVTVSS EIK G2 G2-
EVQLLESGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2A11
SCKASGYTFTTYDLAWVRQA TCRASQDISRWLAWYQQKPG PGQGLEWMGWINPNSGGTN
KAPKLLIYAASRLQAGVPSRF YAQKFQGRVTMTRDTSTSTV SGSGSGTDFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARA TYYCQQSYSTPYSFGQGTKLE NWLDYWGQGTLVTVSS IK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2C06
SCKSSGYSFDSYVVNWVRQA TCRASQTISSWLAWYQQKPG PGQGLEWMGWINPNSGGTN
KAPKLLIYAASSLQSGVPSRF YAQKFQGRVTMTRDTSTSTV SGSGSGTDFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARD TYYCQQSYSTPFTFGPGTKVD WVLDYWGQGTLVTVSS IK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1G01
SCKASGYTFTSYGISWVRQAP TCRASQTISSWLAWYQQKPG GQGLEWMGWMNPNSGGTN
KAPKLLIYAASSLQSGVPSRF YAQKFQGRVTMTRDTSTSTV SGSGSGTDFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARG TYYCQQSYGVPYTFGQGTKV EWLDYWGQGTLVTVSS EIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1C02
SCKASGYTFTSYGISWVRQAP TCRASQSISNWLAWYQQKPG GQGLEWMGWINPNSGGTNY
KAPKLLIYAASSLQSGVPSRF AQKFQGRVTMTRDTSTSTVY SGSGSGTDFTLTISSLQPEDFA
MELSSLRSEDTAVYYCARGW TYYCQQSYSAPYTFGPGTKV ELGYWGQGTLVTVSS DIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQPSSLSASVGDRVTI P1H01
SCKASGYTFTRYTINWVRQA TCRASQSVGNWLAWYQQKP PGQGLEWMGWINPNSGGTN
GKAPKLLIYGASSLQTGVPSR YAQKFQGRVTMTRDTSTSTV FSGSGSGTDFTLTISSLQPEDF
YMELSSLRSEDTAVYYCARD ATYYCQQSYSAPYTFGQGTK FVGYDDWGQGTLVTVSS VEIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1B12
SCKASGYTFTSYGITWVRQAP TCRASQNIGNWLAWYQQKP GQGLEWMGWINPNSGGTNY
GKAPKLLIYAASTLQTGVPSR AQKFQGRVTMTRDTSTSTVY FSGSGSGTDFTLTISSLQPEDF
MELSSLRSEDTAVYYCARDY ATYYCQQSYSAPYSFGQGTK GDLDYWGQGTLVTVSS LEIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1B06
SCKASGGTFSNYILSWVRQAP TCRASQSISRWLAWYQQKPG GQGLEWMGWINPDSGGTNY
KAPKLLIYAASSLQSGVPSRF AQKFQGRVTMTRDTSTSTVY SGSGSGTDFTLTISSLQPEDFA
MELSSLRSEDTAVYYCARGS TYYCQQSYSTPYTFGQGTKL YGMDVWGQGTTVTVSS EIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2H10
SCKASGYSFTRYNMHWVRQ TCRASQSISSWLAWYQQKPG APGQGLEWMGWINPNSGGT
KAPKLLIYGASSLQSGVPSRF NYAQKFQGRVTMTRDTSTST SGSGSGTDFTLTISSLQPEDFA
VYMELSSLRSEDTAVYYCAR TYYCQQSYSVPYSFGQGTKL DGYSGLDVWGKGTTVTVSS EIK
G2 G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1H10
SCKASGGTFSSYAISWVRQAP TCRASQSISKWLAWYQQKPG GQGLEWMGWINPNNGGTNY
KAPKLLIYAASSLQSGVPSRF AQKFQGRVTMTRDTSTSTVY SGSGSGTDFTLTISSLQPEDFA
MELSSLRSEDTAVYYCARDS TYYCQQSYSAPYTFGQGTKV GVGMDVWGQGTTVTVSS EIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2C11
SCKASGGTFNNYAFSWVRQA TCRASQGISNYLAWYQQKPG PGQGLEWMGWINPNSGGTN
KAPKLLIYAASTLQSGVPSRF YAQKFQGRVTMTRDTSTSTV SGSGSGTDFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARD TYYCQQSYSVPYSFGQGTKL GVAVASDYWGQGTLVTVSS EIK
G2 G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1C09
SCKASGYTFSSYNMHWVRQ TCRASQTISNYLNWYQQKPG APGQGLEWMGWINGNTGGT
KAPKLLIYAASNLQSGVPSRF NYAQKFQGRVTMTRDTSTST SGSGSGTDFTLTISSLQPEDFA
VYMELSSLRSEDTAVYYCAR TYYCQQSYSTPQTFGQGTKV GVNVDDFDYWGQGTLVTVS EIK S
G2 G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1A10
SCKASGGTFSSYAFSWVRQA TCRASRDIGRAVGWYQQKPG PGQGLEWMGWINPDTGYTR
KAPKLLIYAASSLQSGVPSRF YAQKFQGRVTMTRDTSTSTV SGSGSGTDFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARG TYYCQQLDSYPFTFGPGTKV DYTGNWYFDLWGRGTLVTV DIK
SS G2 G2- EVQLLESGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1B10
SCKASGYTFTSYGISWVRQAP TCRASQSISSWLAWYQQKPG GQGLEWMGWINPYSGGTNY
KAPKLLIYAASTLQSGVPSRF AQKLQGRVTMTRDTSTSTVY SGSGSGTDFTLTISSLQPEDFA
MELSSLRSEDTAVYYCARAN TYYCQQSYSSPYTFGPGTKV WLDYWGQGTLVTVSS DIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1D07
SCKASGYTFTSYGISWVRQAP TCQASQDISNYLNWYQQKPG GQGLEWMGWISAYNGYTNY
KAPKLLIYAASSLQSGVPSRF AQNLQGRVTMTRDTSTSTVY SGSGSGTDFTLTISSLQPEDFA
MELSSLRSEDTAVYYCARDQ TYYCQQSYSTPLTFGGGTKLE FYGGNSGGHDYWGQGTLVT IK
VSS G2 G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1E05
SCKASGYTFTDYNMHWVRQ TCRASQSIGRWLAWYQQKPG APGQGLEWMGWMNPNSGGT
KAPKLLIYAASSLQSGVPSRF NYAQKFQGRVTMTRDTSTST SGSGSGTDFTLTISSLQPEDFA
VYMELSSLRSEDTAVYYCAR TYYCQQSYSTPYSFGQGTKV E-EDYWGQGTLVTVSS EIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1D03
SCKASGYTFTRYTINWVRQA TCRASQSISTWLAWYQQKPG PGQGLEWMGWINPNSGGAN
KAPKLLIYAASTLQSGVPSRF YAQKFQGRVTMTRDTSTSTV SGSGSGTDFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARG TYYCQQSYSTPYTFAQGTKL DWFDPWGQGTLVTVSS EIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1G12
SCKASGYTFTSYLMHWVRQA TCQASQDISNYLNWYQQKPG PGQGLEWMGWISPNSGGTNY
KAPKLLIYGASRLQSGVPSRF AQKFQGRVTMTRDTSTSTVY SGSGSGTDFTLTISSLQPEDFA
MELSSLRSEDTAVYYCARGD TYYCQQSYSTPYTFGQGTKL WFDPWGQGTLVTVSS EIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2H11
SCKASGYTFSDYYVHWVRQ TCRASQSISSWLAWYQQKPG APGQGLEWMGWINPNSGGT
KAPKLLIYAASTLQSGVPSRF NYAQKFQGRVTMTRDTSTST SGSGSGTDFTLTISSLQPEDFA
VYMELSSLRSEDTAVYYCAR TYYCQQSYSTPFTFGPGTKVD GEWFDPWGQGTLVTVSS IK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1C03
SCKASGYTFTTYYMHWVRQ TCRASQSVSNWLAWYQQKP APGQGLEWMGWINPNSGGT
GKAPKLLIYAASSLQSGVPSR NYAQKFQGRVTMTRDTSTST FSGSGSGTDFTLTISSLQPEDF
VYMELSSLRSEDTAVYYCAR ATYYCQQSYSTPTFGQGTKL SDWFDPWGQGTLVTVSS EIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1G07
SCKASGGTFSNYAINWVRQA TCQASQDISNYLNWYQQKPG PGQGLEWMGWISPYSGGTNY
KAPKLLIYAASTLQSGVPSRF AQKFQGRVTMTRDTSTSTVY SGSGSGTDFTLTISSLQPEDFA
MELSSLRSEDTAVYYCARDS TYYCQQTYAIPLTFGGGTKVE GSYFDYWGQGTLVTVSS IK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1F12
SCKASGYTFTDYYMHWVRQ TCQASQDIGSWLAWYQQKPG APGQGLEWMGWIYPNTGGT
KAPKLLIYATSSLQSGVPSRFS NYAQKFQGRVTMTRDTSTST GSGSGTDFTLTISSLQPEDFAT
VYMELSSLRSEDTAVYYCAR YYCQQSYSTPYTFGQGTKLEI DYGGYVDYWGQGTLVTVSS K G2
G2- EVQLLESGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1G03
SCKASGYTFTSYAMNWVRQ TCRASQGISRWLAWYQQKPG APGQGLEWMGWMNPNSGGT
KAPKLLIYAASTLQPGVPSRF KYAQKFQGRVTMTRDTSTST SGSGSGTDFTLTISSLQPEDFA
VYMELSSLRSEDTAVYYCAR TYYCQQSYIAPFTFGPGTKVD EGPAALDVWGQGTLVTVSS IK
G2 G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2B08
SCKASGYTLTSHLIHWVRQA TCRASQGISNYLAWYQQKPG PGQGLEWMGWINPNSGGTN
KAPKLLIYAASRLESGVPSRF YAQKFQGRVTMTRDTSTSTV SGSGSGTDFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARE TYYCQQSYSIPLTFGGGTKVE RRSGMDVWGQGTTVTVSS IK G2
G2- EVQLLESGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2A10
SCKASGYSFTDYIVHWVRQA TCRASQSISSYLNWYQQKPG PGQGLEWMGWINPYSGGTK
KAPKLLIYGVSSLQSGVPSRF YAQKFQGRVTMTRDTSTSTV SGSGSGTDFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARV TYYCQQSYSNPTFGQGTKVEI LQEGMDVWGQGTLVTVSS K G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2D04
SCKASGYTFSNFLINWVRQAP TCRASQSISSWVAWYQQKPG GQGLEWMGWINPNSGGTNY
KAPKLLIYGASNLESGVPSRF AQKFQGRVTMTRDTSTSTVY SGSGSGTDFTLTISSLQPEDFA
MELSSLRSEDTAVYYCASERE TYYCQQSYSTPYSFGQGTKLE LPFDIWGQGTMVTVSS IK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1C06
SCKASGYTFTDYQMFWVRQ TCRASQGISNYLAWYQQKPG APGQGLEWMGWINPNSGGT
KAPKLLIYAASSLQSGVPSRF NYAQKFQGRVTMTRDTSTST SGSGSGTDFTLTISSLQPEDFA
VYMELSSLRSEDTAVYYCAK TYYCQQSYSDQWTFGQGTK GGGGYGMDVWGQGTTVTVS VEIK S
G2 G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2A09
SCKASGGTFSSYAISWVRQAP TCRASQSISRWLAWYQQKPG GQGLEWMGWINPNSGGTNY
KAPKLLIYAASSLQSGVPSRF AQKFQGRVTMTRDTSTSTVY SGSGSGTDFTLTISSLQPEDFA
MELSSLRSEDTAVYYCAAMG TYYCQQSYLPPYSFGQGTKV IAVAGGMDVWGQGTLVTVS EIK S
G2 G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1B08
SCKASGYTFTNYHMHWVRQ TCRASQSISNWLAWYQQKPG APGQGLEWMGWIHPDSGGTS
KAPKLLIYAASSLQSGVPSRF YAQKFQGRVTMTRDTSTSTV SGSGSGTYFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARN TYYCQQSYSSPYTFGQGTKLE WNLDYWGQGTLVTVSS IK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1E03
SCKASGYTFTGYYMHWVRQ TCRASQSISHYLNWYQQKPG APGQGLEWMGWMNPNSGNT
KAPKLLIYGASSLQSGVPSRF GYAQKFQGRVTMTRDTSTST SGSGSGTDFTLTISSLQPEDFA
VYMELSSLRSEDTAVYYCAT TYYCQQSYTTPWTFGQGTRL YDDGMDVWGQGTTVTVSS EIK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2A03
SCKASGYTFTSYTVNWVRQA TCRASQSISSWLAWYQQKPG PGQGLEWMGWINPNSGGTK
KAPKLLIYAASTLQSGVPSRF YAQNFQGRVTMTRDTSTSTV SGSGSGTDFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARG TYYCQQSYLPPYSFGQGTKLE GGGALDYWGQGTLVTVSS IK G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P2F01
SCKASGYTFTSYYMHWVRQ TCQASQDISNYLNWYQQKPG APGQGLEWMGMINPRDDTT
KAPKLLIYGASRLQSGVPSRF DYARDFQGRVTMTRDTSTST SGSGSGTDFTLTISSLQPEDFA
VYMELSSLRSEDTAVYYCAL TYYCQEGITYTFGQGTKVEIK SGNYYGMDVWGQGTTVTVS S G2
G2- QVQLVQSGAEVKKPGASVKV DIQMTQSPSSLSASVGDRVTI P1H11
SCKASGYTFTNYYMHWVRQ TCQASQDISNYLNWYQQKPG APGQGLEWMGMINPSGGGTS
KAPKLLIYAASSLQSGVPSRF YAQKFQGRVTMTRDTSTSTV SGSGSGTDFTLTISSLQPEDFA
YMELSSLRSEDTAVYYCARG TYYCQQYYSYPFTFGPGTKV NPWELRLDYWGQGTLVTVSS
DIK
G2 G2- QVQLVQSGAEVKKPGSSVKV EIVMTQSPATLSVSPGERATL P1D06
SCKASGYTFTSQYMHWVRQ SCRASQSVSRNLAWYQQKPG APGQGLEWMGRIIPLLGIVNY
QAPRLLIYGASTRATGIPARFS AQKFQGRVTITADESTSTAY GSGSGTEFTLTISSLQSEDFAV
MELSSLRSEDTAVYYCARDK YYCQHYGYSPVTFGQGTKLE NYYGMDVWGQGTTVTVSS IK
TABLE-US-00062 TABLE 34 CDR sequences for G2 selective binders,
selective for HLA-PEPTIDE Target HLA-A*01:01 NTDNNLAVY (determined
according to Kabat numbering) Target Clone group name CDR-H1 CDR-H2
CDR-H3 CDR-L1 CDR-L2 CDR-L3 G2 G2- GTFSSA GWIYPN CAATE RASQSI
AASSLR CQQSYN P2E07 TIS SGGTVY WLGVW STWLA S TPYTF A G2 G2- GTFSSY
GWINPN CARAN RASQSI AASTV CQQSYS P2E03 AIS SGGTIS WLDYW SRWLA QS
TPYTF A G2 G2- YTFTTY GWINPN CARAN RASQDI AASRLQ CQQSYS P2A11 DLA
SGGTNY WLDYW SRWLA A TPYSF A G2 G2- YSFDSY GWINPN CARDW RASQTI
AASSLQ CQQSYS P2C06 VVN SGGTNY VLDYW SSWLA S TPFTF A G2 G2- YTFTSY
GWMNP CARGE RASQTI AASSLQ CQQSYG P1G01 GIS NSGGTN WLDYW SSWLA S
VPYTF YA G2 G2- YTFTSY GWINPN CARGW RASQSI AASSLQ CQQSYS P1C02 GIS
SGGTNY ELGYW SNWLA S APYTF A G2 G2- YTFTRY GWINPN CARDFV RASQSV
GASSLQ CQQSYS P1H01 TIN SGGTNY GYDDW GNWLA T APYTF A G2 G2- YTFTSY
GWINPN CARDY RASQNI AASTLQ CQQSYS P1B12 GIT SGGTNY GDLDY GNWLA T
APYSF A W G2 G2- GTFSNY GWINPD CARGSY RASQSI AASSLQ CQQSYS P1B06
ILS SGGTNY GMDVW SRWLA S TPYTF A G2 G2- YSFTRY GWINPN CARDG RASQSI
GASSLQ CQQSYS P2H10 NMH SGGTNY YSGLDV SSWLA S VPYSF A W G2 G2-
GTFSSY GWINPN CARDSG RASQSI AASSLQ CQQSYS P1H10 AIS NGGTN VGMDV
SKWLA S APYTF YA W G2 G2- GTFNNY GWINPN CARDG RASQGI AASTLQ CQQSYS
P2C11 AFS SGGTNY VAVAS SNYLA S VPYSF A DYW G2 G2- YTFSSY GWING
CARGV RASQTI AASNL CQQSYS P1C09 NMH NTGGT NVDDF SNYLN QS TPQTF NYA
DYW G2 G2- GTFSSY GWINPD CARGD RASRDI AASSLQ CQQLDS P1A10 AFS
TGYTRY YTGNW GRAVG S YPFTF A YFDLW G2 G2- YTFTSY GWINPY CARAN
RASQSI AASTLQ CQQSYS P1B10 GIS SGGTNY WLDYW SSWLA S SPYTF A G2 G2-
YTFTSY GWISAY CARDQF QASQDI AASSLQ CQQSYS P1D07 GIS NGYTN YGGNS
SNYLN S TPLTF YA GGHDY W G2 G2- YTFTDY GWMNP CAREED RASQSI AASSLQ
CQQSYS P1E05 NMH NSGGTN GRWLA S TPYSF YA YW G2 G2- YTFTRY GWINPN
CARGD RASQSI AASTLQ CQQSYS P1D03 TIN SGGAN WFDPW STWLA S TPYTF YA
G2 G2- YTFTSY GWISPN CARGD QASQDI GASRLQ CQQSYS P1G12 LMH SGGTNY
WFDPW SNYLN S TPYTF A G2 G2- YTFSDY GWINPN CARGE RASQSI AASTLQ
CQQSYS P2H11 YVH SGGTNY WFDPW SSWLA S TPFTF A G2 G2- YTFTTY GWINPN
CARSD RASQSV AASSLQ CQQSYS P1C03 YMH SGGTNY WFDPW SNWLA S TPTF A G2
G2- GTFSNY GWISPY CARDSG QASQDI AASTLQ CQQTY P1G07 AIN SGGTNY SYFDY
SNYLN S AIPLTF A W G2 G2- YTFTDY GWIYPN CARDY QASQDI ATSSLQ CQQSYS
P1F12 YMH TGGTN GGYVD GSWLA S TPYTF YA YW G2 G2- YTFTSY GWMNP
CAREGP RASQGI AASTLQ CQQSYI P1G03 AMN NSGGTK AALDV SRWLA P APFTF YA
W G2 G2- YTLTSH GWINPN CARERR RASQGI AASRLE CQQSYS P2B08 LIH SGGTNY
SGMDV SNYLA S IPLTF A W G2 G2- YSFTDY GWINPY CARVL RASQSI GVSSLQ
CQQSYS P2A10 IVH SGGTKY QEGMD SSYLN S NPTF A VW G2 G2- YTFSNF
GWINPN CASERE RASQSI GASNLE CQQSYS P2D04 LIN SGGTNY LPFDIW SSWVA S
TPYSF A G2 G2- YTFTDY GWINPN CAKGG RASQGI AASSLQ CQQSYS P1C06 QMF
SGGTNY GGYGM SNYLA S DQWTF A DVW G2 G2- GTFSSY GWINPN CAAMGI RASQSI
AASSLQ CQQSYL P2A09 AIS SGGTNY AVAGG SRWLA S PPYSF A MDVW G2 G2-
YTFTNY GWIHPD CARNW RASQSI AASSLQ CQQSYS P1B08 HMH SGGTSY NLDYW
SNWLA S SPYTF A G2 G2- YTFTGY GWMNP CATYD RASQSI GASSLQ CQQSYT
P1E03 YMH NSGNTG DGMDV SHYLN S TPWTF YA W G2 G2- YTFTSY GWINPN
CARGG RASQSI AASTLQ CQQSYL P2A03 TVN SGGTKY GGALD SSWLA S PPYSF A
YW G2 G2- YTFTSY GMINPR CALSGN QASQDI GASRLQ CQEGIT P2F01 YMH DDTTD
YYGMD SNYLN S YTF YA VW G2 G2- YTFTNY GMINPS CARGNP QASQDI AASSLQ
CQQYYS P1H11 YMH GGGTSY WELRL SNYLN S YPFTF A DYW G2 G2- YTFTSQ
GRIIPLL CARDK RASQSV GASTRA CQHYG P1D06 YMH GIVNYA NYYGM SRNLA T
YSPVTF DVW
TABLE-US-00063 TABLE 35 VH and VL sequences for scFv selective
binders selective for HLA-PEPTIDE Target HLA-A*02:01 LLASSILCA.
Target Clone group name VH VL G7 G7R3- QVQLVQSGAEVKKPGASVKVS
EIVMTQSPATLSVSPGERATL P1C6 CKASGGTFSNYGISWVRQAPG
SCRASQSVSSSNLAWYQQKPG QGLEWMGIINPGGSTSYAQKF QAPRLLIYGASTRATGIPARF
QGRVTMTRDTSTSTVYMELSS SGSGSGTEFTLTISSLQSEDF LRSEDTAVYYCARDGYDFWSG
AVYYCHHYGRSHTFGQGTKVE YTSDDYWGQGTLVTVSS IK G7 G7R3-
EVQLLESGGGLVQPGGSLRLS DIQMTQSPSSLSASVGDRVTI P1G10
CAASGFTFSSYAMHWVRQAPG TCRASQDIRNDLGWYQQKPGK KGLEWVSGISGSGGSTYYADS
APKLLIYAASSLQSGVPSRFS VKGRFTISRDNSKNTLYLQMN GSGSGTDFTLTISSLQPEDFA
SLRAEDTAVYYCASDYGDYRG TYYCQQANAFPPTFGQGTKVE QGTLVTVSS IK G7 1-G7R3-
QVQLVQSGAEVKKPGASVKVS DIVMTQSPDSLAVSLGERATI P1B4
CKASGYTFSNYYIHWVRQAPG NCKSSQSVFYSSNNKNQLAWY QGLEWMGWLNPNSGNTGYAQR
QQKPGQPPKLLIYWASTRESG FQGRVTMTRDTSTSTVYMELS VPDRFSGSGSGTDFTLTISSL
SLRSEDTAVYYCARDLMTTVV QAEDVAVYYCQQYYSIPLTFG TPGDYGMDVWGQGTTVTVSS
QGTKLEIK G7 2-G7R4- QVQLVQSGAEVKKPGASMKVS DIQMTQSPSSLSASVGDRVTI
P2C2 CKASGYTFTTDGISWVRQAPG TCQASQDIFKYLNWYQQKPGK
QGLEWMGRIYPHSGYTEYAKK APKLLIYAASTLQSGVPSRFS FKGRVTMTRDTSTSTVYMELS
GSGSGTDFTLTISSLQPEDFA SLRSEDTAVYYCARQDGGAFA TYYCQQSYSTPPTFGQGTRLE
FDIWGQGTMVTVSS IK G7 3-G7R4- QVQLVQSGAEVKKPGASVKVS
DIQMTQSPSSLSASVGDRVTI P1A3 CKASGYTFTSQYMHWVRQAPG
TCRASQSISTWLAWYQQKPGK QGLEWMGWISPNNGDTNYAQK APKLLIYYASSLQSGVPSRFS
FQGRVTMTRDTSTSTVYMELS GSGSGTDFTLTISSLQPEDFA SLRSEDTAVYYCARELGYYYG
TYYCQQSYSFPYTFGQGTKVE MDVWGQGTTVTVSS IK G7 4-G7R4-
QVQLVQSGAEVKKPGSSVKVS DIVMTQSPLSLPVTPGEPASI B5-P2E9
CKASRYTFTSYDINWVRQAPG SCSSSQSLLHSNGYNYLDWYL QGLEWMGRIIPMLNIANYAPK
QKPGQSPQLLIYLGSNRASGV FQGRVTITADESTSTAYMELS PDRFSGSGSGTDFTLKISRVE
SLRSEDTAVYYCARALIFGVP AEDVGVYYCMQALQTPLTFGG LLPYGMDVWGQGTTVTVSS
GTKVEIK G7 5-G7R4- EVQLLQSGGGLVQPGGSLRLS DIQMTQSPSSLSASVGDRVTI B10-
CAASGFTFSSSWMHWVRQAPG TCQASQDISNYLNWYQQKPGK P1F8
KGLEWVSFISTSSGYIYYADS APKLLIYSASNLRSGVPSRFS VKGRFTISRDNSKNTLYLQMN
GSGSGTDFTLTISSLQPEDFA SLRAEDTAVYYCAKDLATVGE TYYCQQGNTFPLTFGQGTKVE
PYYYYGMDVWGQGTTVTVSS IK G7 B7 QVQLVQSGAEVKKPGSSVKVS
DIVMTQSPLSLPVTPGEPASI (G7R3- CKASGDTFNTYALSWVRQAPG
SCRSSQSLLHSNGYNYLDWYL P3A9) QGLEWMGWMNPNSGNAGYAQK
QKPGQSPQLLIYLGSNRASGV FQGRVTITADESTSTAYMELS PDRFSGSGSGTDFTLKISRVE
SLRSEDTAVYYCARLWFGELH AEDVGVYYCMQGSHWPPSFGQ YYYYYGMDVWGQGTMVTVSS
GTRLEIK
TABLE-US-00064 TABLE 36 CDR sequences for G7 selective binders
selective for HLA-PEPTIDE Target HLA-A*02:01 LLASSILCA Target Clone
group name CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 G7 G7R3-
GTFSNY GIINPG CARDGY RASQSV GASTRAT CHHYGR PIC6 GIS GSTSYA DFWSGY
SSSNLA SHTF TSDDYW G7 G7R3- FTFSSY SGISGS CASDYG RASQDI AASSLQS
CQQANA P1G10 AMH GGSTYY DYR RNDLG FPPTF A G7 1-G7R3- YTFSNY GWLNPN
CARDLM KSSQSV WASTRES CQQYYS P1B4 YIH SGNTGY TTVVTP FYSSNN IPLTF A
GDYGMD KNQLA VW G7 2-G7R4- YTFTTD GRIYPH CARQDG QASQDI AASTLQS
CQQSYS P2C2 GIS SGYTEY GAFAFD FKYLN TPPTF A IW G7 3-G7R4- YTFTSQ
GWISPN CARELG RASQSI YASSLQS CQQSYS P1A3 YMH NGDTNY YYYGMD STWLA
FPYTF A VW G7 4-G7R4- YTFTSY GRIIPM CARALI SSSQSL LGSNRAS CMQALQ
B5-P2E9 DIN LNIANY FGVPLL LHSNGY TPLTF A PYGMDV NYLD W G7 5-G7R4-
FTFSSS SFISTS CAKDLA QASQDI SASNLRS CQQGNT B10-P1F8 WMH SGYIYY
TVGEPY SNYLN FPLTF A YYYGMD VW G7 B7(G7R3- DTFNTY GWMNPN CARLWF
RSSQSL LGSNRAS CMQGSH P3A9) ALS SGNAGY GELHYY LHSNGY WPPSF A YYYGMD
NYLD VW
TABLE-US-00065 TABLE 38 amino acid sequences of selected HLA
subtypes and B2MG (beta-2 microglobulin) A*01:01
MGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQKMEPRA
PWIEQEGPEYWDQETRNMKAHSQTDRANLGTLRGYYNQSEDGSHTIQIMY
GCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEA
VHAAEQRRVYLEGRCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEA
TLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVV
PSGEEQRYTCHVQHEGLPKPLTLR A*02:01
MGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVREDSDAASQRMEPRA
PWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMY
GCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEA
AHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEA
TLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVV
PSGQEQRYTCHVQHEGLPKPLTLR B*35:01
MGSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVREDSDAASPRTEPRA
PWIEQEGPEYWDRNTQIFKTNTQTYRESLRNLRGYYNQSEAGSHIIQRMY
GCDLGPDGRLLRGHDQSAYDGKDYIALNEDLSSWTAADTAAQITQRKWEA
ARVAEQLRAYLEGLCVEWLRRYLENGKETLQRADPPKTHVTHHPVSDHEA
TLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVV
PSGEEQRYTCHVQHEGLPKPLTLR B2MG
MIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKV
EHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM
[1064] CDR3 and V(D)J Sequences of TCR Clonotypes Confirmed Through
Resorting
[1065] These sequences are included in PCT/US2018/046997, filed on
Aug. 17, 2018, which application is incorporated by reference in
its entirety.
[1066] CDR3 and V(D)J Sequences of TCR Clonotypes Confirmed Through
Cloning
[1067] These sequences are included in PCT/US2018/046997, filed on
Aug. 17, 2018, which application is incorporated by reference in
its entirety.
Table A
[1068] This table is included in PCT/US2018/06793, filed on Dec.
28, 2018, which is incorporated by reference in its entirety
Table A1
[1069] This table is included in PCT/US2018/046997, filed on Aug.
17, 2018, which is incorporated by reference in its entirety.
Table A2
[1070] This table is included in PCT/US2018/046997, filed on Aug.
17, 2018, which is incorporated by reference in its entirety.
Sequence CWU 1
1
6651274PRTHomo sapiens 1Met Gly Ser His Ser Met Arg Tyr Phe Tyr Thr
Ala Met Ser Arg Pro1 5 10 15Gly Arg Gly Glu Pro Arg Phe Ile Ala Val
Gly Tyr Val Asp Asp Thr 20 25 30Gln Phe Val Arg Phe Asp Ser Asp Ala
Ala Ser Pro Arg Thr Glu Pro 35 40 45Arg Ala Pro Trp Ile Glu Gln Glu
Gly Pro Glu Tyr Trp Asp Arg Asn 50 55 60Thr Gln Ile Phe Lys Thr Asn
Thr Gln Thr Tyr Arg Glu Ser Leu Arg65 70 75 80Asn Leu Arg Gly Tyr
Tyr Asn Gln Ser Glu Ala Gly Ser His Ile Ile 85 90 95Gln Arg Met Tyr
Gly Cys Asp Leu Gly Pro Asp Gly Arg Leu Leu Arg 100 105 110Gly His
Asp Gln Ser Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn 115 120
125Glu Asp Leu Ser Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Thr
130 135 140Gln Arg Lys Trp Glu Ala Ala Arg Val Ala Glu Gln Leu Arg
Ala Tyr145 150 155 160Leu Glu Gly Leu Cys Val Glu Trp Leu Arg Arg
Tyr Leu Glu Asn Gly 165 170 175Lys Glu Thr Leu Gln Arg Ala Asp Pro
Pro Lys Thr His Val Thr His 180 185 190His Pro Val Ser Asp His Glu
Ala Thr Leu Arg Cys Trp Ala Leu Gly 195 200 205Phe Tyr Pro Ala Glu
Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp 210 215 220Gln Thr Gln
Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Arg225 230 235
240Thr Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln
245 250 255Arg Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro
Leu Thr 260 265 270Leu Arg29PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 2Glu Val Asp Pro Ile Gly His
Val Tyr1 5314PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 3Cys Ala Arg Asp Gly Val Arg Tyr Tyr Gly
Met Asp Val Trp1 5 10415PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 4Cys Ala Arg Gly Val Arg Gly
Tyr Asp Arg Ser Ala Gly Tyr Trp1 5 10 15516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Cys
Ala Ser His Asp Tyr Gly Asp Tyr Gly Glu Tyr Phe Gln His Trp1 5 10
15621PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Cys Ala Arg Val Ser Trp Tyr Cys Ser Ser Thr Ser
Cys Gly Val Asn1 5 10 15Trp Phe Asp Pro Trp 20715PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Cys
Ala Lys Val Asn Trp Asn Asp Gly Pro Tyr Phe Asp Tyr Trp1 5 10
15821PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Cys Ala Thr Pro Thr Asn Ser Gly Tyr Tyr Gly Pro
Tyr Tyr Tyr Tyr1 5 10 15Gly Met Asp Val Trp 2099PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Cys
Ala Arg Asp Val Met Asp Val Trp1 51011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Cys
Ala Arg Glu Gly Tyr Gly Met Asp Val Trp1 5 101112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Cys
Ala Arg Asp Asn Gly Val Gly Val Asp Tyr Trp1 5 101225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Cys
Ala Arg Gly Ile Ala Asp Ser Gly Ser Tyr Tyr Gly Asn Gly Arg1 5 10
15Asp Tyr Tyr Tyr Gly Met Asp Val Trp 20 251311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Cys
Ala Arg Gly Asp Tyr Tyr Phe Asp Tyr Trp1 5 101414PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Cys
Ala Arg Asp Gly Thr Arg Tyr Tyr Gly Met Asp Val Trp1 5
101512PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Cys Ala Arg Asp Val Val Ala Asn Phe Asp Tyr
Trp1 5 101618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 16Cys Ala Arg Gly His Ser Ser Gly Trp
Tyr Tyr Tyr Tyr Gly Met Asp1 5 10 15Val Trp1713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Cys
Ala Lys Asp Leu Gly Ser Tyr Gly Gly Tyr Tyr Trp1 5
101819PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Cys Ala Arg Ser Trp Phe Gly Gly Phe Asn Tyr His
Tyr Tyr Gly Met1 5 10 15Asp Val Trp1914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Cys
Ala Arg Glu Leu Pro Ile Gly Tyr Gly Met Asp Val Trp1 5
102015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Cys Ala Arg Gly Gly Ser Tyr Tyr Tyr Tyr Gly Met
Asp Val Trp1 5 10 152111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 21Cys Met Gln Gly Leu Gln Thr
Pro Ile Thr Phe1 5 102211PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Cys Met Gln Ala Leu Gln Thr
Pro Pro Thr Phe1 5 102311PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 23Cys Gln Gln Ala Ile Ser Phe
Pro Leu Thr Phe1 5 102411PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 24Cys Gln Gln Ala Asn Ser Phe
Pro Leu Thr Phe1 5 102511PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 25Cys Gln Gln Ser Tyr Ser Ile
Pro Leu Thr Phe1 5 102611PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 26Cys Gln Gln Thr Tyr Met Met
Pro Tyr Thr Phe1 5 102711PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 27Cys Gln Gln Ser Tyr Ile Thr
Pro Trp Thr Phe1 5 102811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 28Cys Gln Gln Ser Tyr Ile Thr
Pro Tyr Thr Phe1 5 102911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 29Cys Gln Gln Tyr Tyr Thr Thr
Pro Tyr Thr Phe1 5 103011PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 30Cys Gln Gln Ser Tyr Ser Thr
Pro Leu Thr Phe1 5 103111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 31Cys Met Gln Ala Leu Gln Thr
Pro Leu Thr Phe1 5 103211PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 32Cys Gln Gln Tyr Gly Ser Trp
Pro Arg Thr Phe1 5 103311PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 33Cys Gln Gln Ser Tyr Ser Thr
Pro Val Thr Phe1 5 103411PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 34Cys Met Gln Ala Leu Gln Thr
Pro Tyr Thr Phe1 5 103511PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 35Cys Gln Gln Ala Asn Ser Phe
Pro Phe Thr Phe1 5 1036119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 36Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asp Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile
Asn Pro Arg Ser Gly Ser Thr Lys Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Gly Val Arg Tyr Tyr Gly Met Asp Val Trp Gly Gln
Gly 100 105 110Thr Thr Val Thr Val Ser Ser 11537120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
37Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
His 20 25 30Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Met Asn Pro Asn Ser Gly Asp Thr Gly Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Val Arg Gly Tyr Asp Arg
Ser Ala Gly Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Ile Val Ser
Ser 115 12038121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 38Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Ser Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ser Tyr Ile Ser Gly
Asp Ser Gly Tyr Thr Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Ser His Asp Tyr Gly Asp Tyr Gly Glu Tyr Phe Gln His Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115 12039126PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
39Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Ser 20 25 30Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Ser Trp Tyr Cys Ser Ser
Thr Ser Cys Gly Val Asn Trp 100 105 110Phe Asp Pro Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 12540120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
40Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Ser 20 25 30Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Ser Ile Ser Ser Ser Gly Gly Tyr Ile Asn Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Asn Trp Asn Asp Gly Pro
Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 12041126PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 41Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Phe Ser Asn Phe 20 25 30Gly Val Ser Trp Leu Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro
Ile Leu Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Thr Pro Thr Asn Ser Gly Tyr Tyr Gly Pro Tyr Tyr Tyr Tyr Gly 100 105
110Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
12542114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 42Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly
Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Val
Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110Ser
Ser43116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 43Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Gly Tyr 20 25 30Leu Val Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly
Gly Thr Asn Thr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly
Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val 100 105 110Thr Val
Ser Ser 11544117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 44Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Ile Phe Arg Asn Tyr 20 25 30Pro Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro
Asp Ser Gly Gly Thr Lys Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asp Asn Gly Val Gly Val Asp Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser 11545130PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 45Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Met
Asn Pro Asn Ile Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ile Ala Asp Ser Gly Ser Tyr Tyr Gly Asn Gly Arg
Asp 100 105 110Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val 115 120 125Ser Ser 13046116PRTArtificial
SequenceDescription of Artificial Sequence
Synthetic polypeptide 46Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly
Val Thr Lys Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Asp
Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val
Ser Ser 11547119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 47Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asp Ile Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro
Asn Ser Gly Asp Thr Lys Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asp Gly Thr Arg Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly 100 105
110Thr Thr Val Thr Val Ser Ser 11548117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
48Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Ser Ser Tyr Thr Asn Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Val Val Ala Asn Phe Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11549123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 49Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Met Asn Pro Asp Ser Gly
Ser Thr Gly Tyr Ala Gln Arg Phe 50 55 60Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly His
Ser Ser Gly Trp Tyr Tyr Tyr Tyr Gly Met Asp Val 100 105 110Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 12050118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
50Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser
Tyr 20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Thr Ser Phe Thr Asn Thr Met Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp Leu Gly Ser Tyr Gly Gly
Tyr Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
11551124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 51Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Gly Gly
Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Trp
Phe Gly Gly Phe Asn Tyr His Tyr Tyr Gly Met Asp 100 105 110Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 12052119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
52Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Leu Pro Ile Gly Tyr Gly
Met Asp Val Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
11553120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 53Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Val Gly
Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly
Ser Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110Gly Thr
Thr Val Thr Val Ser Ser 115 12054112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
54Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Tyr Arg Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Gly 85 90 95Leu Gln Thr Pro Ile Thr Phe Gly Gln
Gly Thr Arg Leu Glu Ile Lys 100 105 11055112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
55Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Ser Arg Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr Pro Pro Thr Phe Gly Pro
Gly Thr Lys Val Asp Ile Lys 100 105 11056107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
56Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ala Ile Ser Phe Pro Leu 85 90 95Thr Phe Gly Gln Ser Thr Lys Val Glu
Ile Lys 100 10557107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 57Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10558107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
58Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ala Asn Ser Phe Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 10559107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 59Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Leu 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10560107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
60Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Tyr Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Thr Tyr Met Met Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10561107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 61Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ile Thr Pro Trp 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10562107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
62Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ile Thr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 10563113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 63Asp Ile Val Met Thr Gln Ser Pro
Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys
Lys Thr Ser Gln Ser Val Leu Tyr Arg 20 25 30Pro Asn Asn Glu Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu
Ile Tyr Gln Ala Ser Ile Arg Glu Pro Gly Val 50 55 60Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95Tyr
Tyr Thr Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105
110Lys64107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 64Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Ser Arg Phe 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser Arg Pro Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 10565112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
65Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr Pro Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11066107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 66Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ala Arg Ala Ser
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Tyr Gly Ser Trp Pro Arg 85 90 95Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 10567107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
67Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Thr Pro Val 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10568112PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 68Asp Ile Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu
Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile
Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95Leu
Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
11069107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 69Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser
Glu Asp Ile Ser Asn His 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Leu Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Phe 85 90 95Thr Phe Gly Pro
Gly Thr Lys Val Asp Ile Lys 100 10570112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
70Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr Pro Leu Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 11071107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
71Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 10572274PRTHomo sapiens 72Met Gly Ser His Ser Met Arg
Tyr Phe Phe Thr Ser Val Ser Arg Pro1 5 10 15Gly Arg Gly Glu Pro Arg
Phe Ile Ala Val Gly Tyr Val Asp Asp Thr 20 25 30Gln Phe Val Arg Phe
Asp Ser Asp Ala Ala Ser Gln Lys Met Glu Pro 35 40 45Arg Ala Pro Trp
Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gln Glu 50 55 60Thr Arg Asn
Met Lys Ala His Ser Gln Thr Asp Arg Ala Asn Leu Gly65 70 75 80Thr
Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Ile 85 90
95Gln Ile Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Phe Leu Arg
100 105 110Gly Tyr Arg Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala
Leu Asn 115 120 125Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala
Ala Gln Ile Thr 130 135 140Lys Arg Lys Trp Glu Ala Val His Ala Ala
Glu Gln Arg Arg Val Tyr145 150 155 160Leu Glu Gly Arg Cys Val Asp
Gly Leu Arg Arg Tyr Leu Glu Asn Gly 165 170 175Lys Glu Thr Leu Gln
Arg Thr Asp Pro Pro Lys Thr His Met Thr His 180 185 190His Pro Ile
Ser Asp His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly 195 200 205Phe
Tyr Pro Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp 210 215
220Gln Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp
Gly225 230 235 240Thr Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser
Gly Glu Glu Gln 245 250 255Arg Tyr Thr Cys His Val Gln His Glu Gly
Leu Pro Lys Pro Leu Thr 260 265 270Leu Arg739PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 73Asn
Thr Asp Asn Asn Leu Ala Val Tyr1 57410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Cys
Ala Ala Thr Glu Trp Leu Gly Val Trp1 5 107510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 75Cys
Ala Arg Ala Asn Trp Leu Asp Tyr Trp1 5 107610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Cys
Ala Arg Asp Trp Val Leu Asp Tyr Trp1 5 107710PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 77Cys
Ala Arg Gly Glu Trp Leu Asp Tyr Trp1 5 107810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 78Cys
Ala Arg Gly Trp Glu Leu Gly Tyr Trp1 5 107911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 79Cys
Ala Arg Asp Phe Val Gly Tyr Asp Asp Trp1 5 108011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Cys
Ala Arg Asp Tyr Gly Asp Leu Asp Tyr Trp1 5 108111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 81Cys
Ala Arg Gly Ser Tyr Gly Met Asp Val Trp1 5 108212PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 82Cys
Ala Arg Asp Gly Tyr Ser Gly Leu Asp Val Trp1 5 108312PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 83Cys
Ala Arg Asp Ser Gly Val Gly Met Asp Val Trp1 5 108413PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84Cys
Ala Arg Asp Gly Val Ala Val Ala Ser Asp Tyr Trp1 5
108513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 85Cys Ala Arg Gly Val Asn Val Asp Asp Phe Asp Tyr
Trp1 5 108615PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 86Cys Ala Arg Gly Asp Tyr Thr Gly Asn
Trp Tyr Phe Asp Leu Trp1 5 10 158717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 87Cys
Ala Arg Asp Gln Phe Tyr Gly Gly Asn Ser Gly Gly His Asp Tyr1 5 10
15Trp888PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 88Cys Ala Arg Glu Glu Asp Tyr Trp1
58910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 89Cys Ala Arg Gly Asp Trp Phe Asp Pro Trp1 5
109010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 90Cys Ala Arg Gly Glu Trp Phe Asp Pro Trp1 5
109110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 91Cys Ala Arg Ser Asp Trp Phe Asp Pro Trp1 5
109212PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 92Cys Ala Arg Asp Ser Gly Ser Tyr Phe Asp Tyr
Trp1 5 109312PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 93Cys Ala Arg Asp Tyr Gly Gly Tyr Val
Asp Tyr Trp1 5 109412PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 94Cys Ala Arg Glu Gly Pro Ala
Ala Leu Asp Val Trp1 5 109512PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 95Cys Ala Arg Glu Arg Arg Ser
Gly Met Asp Val Trp1 5 109612PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 96Cys Ala Arg Val Leu Gln Glu
Gly Met Asp Val Trp1 5 109712PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 97Cys Ala Ser Glu Arg Glu Leu
Pro Phe Asp Ile Trp1 5 109813PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 98Cys Ala Lys Gly Gly Gly Gly
Tyr Gly Met Asp Val Trp1 5 109915PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 99Cys Ala Ala Met Gly Ile
Ala Val Ala Gly Gly Met Asp Val Trp1 5 10 1510010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 100Cys
Ala Arg Asn Trp Asn Leu Asp Tyr Trp1 5 1010111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 101Cys
Ala Thr Tyr Asp Asp Gly Met Asp Val Trp1 5 1010212PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 102Cys
Ala Arg Gly Gly Gly Gly Ala Leu Asp Tyr Trp1 5 1010313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 103Cys
Ala Leu Ser Gly Asn Tyr Tyr Gly Met Asp Val Trp1 5
1010414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 104Cys Ala Arg Gly Asn Pro Trp Glu Leu Arg Leu
Asp Tyr Trp1 5 1010513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 105Cys Ala Arg Asp Lys Asn
Tyr Tyr Gly Met Asp Val Trp1 5 1010611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 106Cys
Gln Gln Ser Tyr Asn Thr Pro Tyr Thr Phe1 5 1010711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 107Cys
Gln Gln Ser Tyr Ser Thr Pro Tyr Thr Phe1 5 1010811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 108Cys
Gln Gln Ser Tyr Ser Thr Pro Tyr Ser Phe1 5 1010911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 109Cys
Gln Gln Ser Tyr Ser Thr Pro Phe Thr Phe1 5 1011011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 110Cys
Gln Gln Ser Tyr Gly Val Pro Tyr Thr Phe1 5 1011111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 111Cys
Gln Gln Ser Tyr Ser Ala Pro Tyr Thr Phe1 5 1011211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 112Cys
Gln Gln Ser Tyr Ser Ala Pro Tyr Ser Phe1 5 1011311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 113Cys
Gln Gln Ser Tyr Ser Val Pro Tyr Ser Phe1 5 1011411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 114Cys
Gln Gln Ser Tyr Ser Thr Pro Gln Thr Phe1 5 1011511PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 115Cys
Gln Gln Leu Asp Ser Tyr Pro Phe Thr Phe1 5 1011611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 116Cys
Gln Gln Ser Tyr Ser Ser Pro Tyr Thr Phe1 5 1011710PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 117Cys
Gln Gln Ser Tyr Ser Thr Pro Thr Phe1 5 1011811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 118Cys
Gln Gln Thr Tyr Ala Ile Pro Leu Thr Phe1 5 1011911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 119Cys
Gln Gln Ser Tyr Ile Ala Pro Phe Thr Phe1 5 1012010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 120Cys
Gln Gln Ser Tyr Ser Asn Pro Thr Phe1 5 1012111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 121Cys
Gln Gln Ser Tyr Ser Asp Gln Trp Thr Phe1 5 1012211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 122Cys
Gln Gln Ser Tyr Leu Pro Pro Tyr Ser Phe1 5 1012311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 123Cys
Gln Gln Ser Tyr Thr Thr Pro Trp Thr Phe1 5 101249PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 124Cys
Gln Glu Gly Ile Thr Tyr Thr Phe1 512511PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 125Cys
Gln Gln Tyr Tyr Ser Tyr Pro Phe Thr Phe1 5 1012611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 126Cys
Gln His Tyr Gly Tyr Ser Pro Val Thr Phe1 5 10127119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
127Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Met Ile Asn Pro Ser Gly Gly Gly Thr Ser Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Asn Pro Trp Glu Leu Arg
Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115128115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 128Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Ala 20 25 30Thr Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Tyr Pro Asn
Ser Gly Gly Thr Val Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Thr Glu Trp Leu Gly Val Trp Gly Gln Gly Thr Thr Val Thr 100 105
110Val Ser Ser 115129115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 129Glu Val Gln Leu Leu
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Pro Asn Ser Gly Gly Thr Ile Ser Ala Pro Asn Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ala Asn Trp Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110Val Ser Ser 115130115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
130Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr
Tyr 20 25 30Asp Leu Ala Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Asn Trp Leu Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser
115131115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 131Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ser Ser
Gly Tyr Ser Phe Asp Ser Tyr 20 25 30Val Val Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser
Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Trp Val Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val
Ser Ser 115132115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 132Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Met Asn Pro
Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Glu Trp Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser 115133115PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 133Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Trp Glu Leu Gly Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110Val Ser Ser 115134116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
134Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg
Tyr 20 25 30Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Phe Val Gly Tyr Asp Asp
Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115135116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 135Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile Thr Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser
Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Tyr Gly Asp Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr
Val Ser Ser 115136116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 136Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr 20 25 30Ile Leu Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Ser Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
Val 100 105 110Thr Val Ser Ser 115137117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
137Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Arg
Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Tyr Ser Gly Leu Asp
Val Trp Gly Lys Gly Thr Thr 100 105 110Val Thr Val Ser Ser
115138117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 138Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Asn
Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Ser Gly Val Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105 110Val
Thr Val Ser Ser 115139118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 139Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Tyr 20 25 30Ala Phe Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Gly Val Ala Val Ala Ser Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 115140118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
140Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser
Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Gly Asn Thr Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Val Asn Val Asp Asp Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
115141120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 141Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Phe Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asp Thr
Gly Tyr Thr Arg Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Asp Tyr Thr Gly Asn Trp Tyr Phe Asp Leu Trp Gly Arg 100 105 110Gly
Thr Leu Val Thr Val Ser Ser 115 120142115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
142Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Tyr Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Leu 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Asn Trp Leu Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser
115143122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 143Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Ser Ala Tyr Asn
Gly Tyr Thr Asn Tyr Ala Gln Asn Leu 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gln Phe Tyr Gly Gly Asn Ser Gly Gly His Asp Tyr Trp 100 105 110Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120144113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
144Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Met Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Glu Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser 100 105 110Ser145115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
145Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg
Tyr 20 25 30Thr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Ala Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Asp Trp Phe Asp Pro Trp
Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser
115146115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 146Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Leu Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Ser Pro Asn Ser
Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Asp Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val
Ser Ser 115147115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 147Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30Tyr Val His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Glu Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val
Ser Ser 115148115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 148Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Ser Asp Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser 115149117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 149Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr 20 25 30Ala Ile Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Ser Pro Tyr Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Ser Gly Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser 115150117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
150Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Tyr Pro Asn Thr Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Tyr Gly Gly Tyr Val Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115151117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 151Glu Val Gln Leu Leu Glu Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Met Asn Pro Asn Ser
Gly Gly Thr Lys Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu
Gly Pro Ala Ala Leu Asp Val Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 115152117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 152Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ser His 20 25 30Leu Ile His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Arg Arg Ser Gly Met Asp Val Trp Gly Gln Gly Thr
Thr 100 105 110Val Thr Val Ser Ser 115153117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
153Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp
Tyr 20 25 30Ile Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Tyr Ser Gly Gly Thr Lys Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Gln Glu Gly Met Asp
Val Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115154117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 154Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Ser Asn Phe 20 25 30Leu Ile Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser
Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Glu
Arg Glu Leu Pro Phe Asp Ile Trp Gly Gln Gly Thr Met 100 105 110Val
Thr Val Ser Ser 115155118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 155Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Gln Met Phe
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Gly Gly Gly Gly Tyr Gly Met Asp Val Trp Gly Gln Gly
Thr 100 105 110Thr Val Thr Val Ser Ser 115156120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
156Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Met Gly Ile Ala Val Ala Gly
Gly Met Asp Val Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120157115PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 157Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30His Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile His Pro
Asp Ser Gly Gly Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asn Trp Asn Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser 115158116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 158Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Thr Tyr Asp Asp Gly Met Asp Val Trp Gly Gln Gly Thr Thr
Val 100 105 110Thr Val Ser Ser 115159117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
159Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Thr Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Lys Tyr Ala
Gln Asn Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Gly Gly Ala Leu Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115160118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 160Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Met Ile Asn Pro Arg Asp
Asp Thr Thr Asp Tyr Ala Arg Asp Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Leu Ser
Gly Asn Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr 100 105 110Thr
Val Thr Val Ser Ser 115161118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 161Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Gln 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg
Ile Ile Pro Leu Leu Gly Ile Val Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Lys Asn Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly
Thr 100 105 110Thr Val Thr Val Ser Ser 115162107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
162Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Tyr Ser Tyr Pro Phe 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys 100 105163107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 163Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asn Thr Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105164107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 164Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Val Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105165107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
165Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Arg
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Arg Leu Gln Ala Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Thr Pro Tyr 85 90 95Ser Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105166107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 166Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe
85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100
105167107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 167Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Ser Trp
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Tyr Gly Val Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105168107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 168Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Asn Trp 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Tyr 85 90 95Thr
Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105169107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
169Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Gly Asn
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Ser Leu Gln Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Ala Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105170107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 170Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asn Ile Gly Asn Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Thr Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Tyr
85 90 95Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105171107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 171Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105172107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
172Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Val Pro Tyr 85 90 95Ser Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105173107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 173Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Lys Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105174107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 174Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Val Pro Tyr 85 90 95Ser Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105175107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
175Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Thr Pro Gln 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105176107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 176Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Arg Asp Ile Gly Arg Ala 20 25 30Val Gly Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asp Ser Tyr Pro Phe
85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100
105177107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 177Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ser Pro Tyr 85 90 95Thr Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys 100 105178107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
178Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105179107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 179Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Arg Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr
85 90 95Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105180107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 180Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Thr Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr 85 90 95Thr Phe Ala
Gln Gly Thr Lys Leu Glu Ile Lys 100 105181107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
181Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Thr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105182107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 182Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe
85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100
105183106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 183Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Val Ser Asn Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Thr 85 90 95Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105184107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
184Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Thr Tyr Ala Ile Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105185107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 185Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105186107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 186Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln
Pro Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ile Ala Pro Phe 85 90 95Thr Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys 100 105187107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
187Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Arg Leu Glu Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Ile Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105188106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 188Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Val Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Tyr Ser Asn Pro Thr 85 90 95Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105189107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
189Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Trp 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Thr Pro Tyr 85 90 95Ser Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105190107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 190Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Asp Gln Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105191107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 191Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Leu Pro Pro Tyr 85 90 95Ser Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105192107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
192Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Ser Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105193107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 193Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser His Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
105194107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 194Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Leu Pro Pro Tyr 85 90 95Ser Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105195105PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
195Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Glu
Gly Ile Thr Tyr Thr Phe 85 90 95Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105196107PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 196Glu Ile Val Met Thr Gln Ser Pro
Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Arg Asn 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asp
Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Tyr Ser Pro Val 85 90 95Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105197274PRTHomo
sapiens 197Met Gly Ser His Ser Met Arg Tyr Phe Phe Thr Ser Val Ser
Arg Pro1 5 10 15Gly Arg Gly Glu Pro Arg Phe Ile Ala Val Gly Tyr Val
Asp Asp Thr 20 25 30Gln Phe Val Arg Phe Asp Ser Asp Ala Ala Ser Gln
Arg Met Glu Pro 35 40 45Arg Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu
Tyr Trp Asp Gly Glu 50 55 60Thr Arg Lys Val Lys Ala His Ser Gln Thr
His Arg Val Asp Leu Gly65 70 75 80Thr Leu Arg Gly Tyr Tyr Asn Gln
Ser Glu Ala Gly Ser His Thr Val 85 90 95Gln Arg Met Tyr Gly Cys Asp
Val Gly Ser Asp Trp Arg Phe Leu Arg 100 105 110Gly Tyr His Gln Tyr
Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys 115 120 125Glu Asp Leu
Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Thr Thr 130 135 140Lys
His Lys Trp Glu Ala Ala His Val Ala Glu Gln Leu Arg Ala Tyr145 150
155 160Leu Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr Leu Glu Asn
Gly 165 170 175Lys Glu Thr Leu Gln Arg Thr Asp Ala Pro Lys Thr His
Met Thr His 180 185 190His Ala Val Ser Asp His Glu Ala Thr Leu Arg
Cys Trp Ala Leu Ser 195 200 205Phe Tyr Pro Ala Glu Ile Thr Leu Thr
Trp Gln Arg Asp Gly Glu Asp 210 215 220Gln Thr Gln Asp Thr Glu Leu
Val Glu Thr Arg Pro Ala Gly Asp Gly225 230 235 240Thr Phe Gln Lys
Trp Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln 245 250 255Arg Tyr
Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro Leu Thr 260 265
270Leu Arg19810PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 198Ala Ile Phe Pro Gly Ala Val Pro Ala
Ala1 5 10199121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 199Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Leu Ser Ser Tyr 20 25 30Pro Ile Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Ser Thr
Tyr Ser Gly His Ala Asp Tyr Ala Gln Lys Leu 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Ser Tyr Asp Tyr Gly Asp Tyr Leu Asn Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115 120200107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
200Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Ile Pro Pro 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Asp
Ile Lys 100 10520116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 201Cys Ala Arg Asp Asp Tyr Gly Asp Tyr
Val Ala Tyr Phe Gln His Trp1 5 10 1520214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 202Cys
Ala Arg Asp Leu Ser Tyr Tyr Tyr Gly Met Asp Val Trp1 5
1020317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 203Cys Ala Arg Val Tyr Asp Phe Trp Ser Val Leu
Ser Gly Phe Asp Ile1 5 10 15Trp20419PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 204Cys
Ala Arg Val Glu Gln Gly Tyr Asp Ile Tyr Tyr Tyr Tyr Tyr Met1 5 10
15Asp Val Trp20516PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 205Cys Ala Arg Ser Tyr Asp Tyr Gly Asp
Tyr Leu Asn Phe Asp Tyr Trp1 5 10 1520618PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 206Cys
Ala Arg Ala Ser Gly Ser Gly Tyr Tyr Tyr Tyr Tyr Gly Met Asp1 5 10
15Val Trp20713PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 207Cys Ala Ala Ser Thr Trp Ile Gln Pro
Phe Asp Tyr Trp1 5 1020817PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 208Cys Ala Ser Asn Gly Asn
Tyr Tyr Gly Ser Gly Ser Tyr Tyr Asn Tyr1 5 10
15Trp20917PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 209Cys Ala Arg Ala Val Tyr Tyr Asp Phe Trp Ser
Gly Pro Phe Asp Tyr1 5 10 15Trp21016PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 210Cys
Ala Lys Gly Gly Ile Tyr Tyr Gly Ser Gly Ser Tyr Pro Ser Trp1 5 10
1521111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 211Cys Ala Arg Gly Leu Tyr Tyr Met Asp Val Trp1 5
1021218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 212Cys Ala Arg Gly Leu Tyr Gly Asp Tyr Phe Leu
Tyr Tyr Gly Met Asp1 5 10 15Val Trp21319PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 213Cys
Ala Arg Gly Leu Leu Gly Phe Gly Glu Phe Leu Thr Tyr Gly Met1 5 10
15Asp Val Trp21419PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 214Cys Ala Arg Asp Arg Asp Ser Ser Trp
Thr Tyr Tyr Tyr Tyr Gly Met1 5 10 15Asp Val Trp21521PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 215Cys
Ala Arg Gly Asp Tyr Tyr Asp Ser Ser Gly Tyr Tyr Phe Pro Val1 5 10
15Tyr Phe Asp Tyr Trp 2021619PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 216Cys Ala Lys Asp Pro Phe
Trp Ser Gly His Tyr Tyr Tyr Tyr Gly Met1 5 10 15Asp Val
Trp21710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 217Cys Gln Gln Asn Tyr Asn Ser Val Thr Phe1 5
1021811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 218Cys Gln Gln Ser Tyr Asn Thr Pro Trp Thr Phe1 5
1021911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 219Cys Gly Gln Ser Tyr Ser Thr Pro Pro Thr Phe1 5
1022011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 220Cys Gln Gln Ser Tyr Ser Ile Pro Pro Thr Phe1 5
1022111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 221Cys Gln Gln His Asn Ser Tyr Pro Pro Thr Phe1 5
1022211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 222Cys Gln Gln Tyr Ser Thr Tyr Pro Ile Thr Ile1 5
1022311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 223Cys Gln Gln Ala Asn Ser Phe Pro Trp Thr Phe1 5
1022411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 224Cys Gln Gln Ser His Ser Thr Pro Gln Thr Phe1 5
1022511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 225Cys Gln Gln Thr Tyr Ser Thr Pro Trp Thr Phe1 5
1022611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 226Cys Gln Gln Tyr Gly Ser Ser Pro Tyr Thr Phe1 5
1022711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 227Cys Gln Gln Ser His Ser Thr Pro Leu Thr Phe1 5
1022811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 228Cys Gln Gln Ala Asn Gly Phe Pro Leu Thr Phe1 5
10229121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 229Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Arg Ser 20 25 30Ala Ile Thr Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser
Gly Ala Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Asp Tyr Gly Asp Tyr Val Ala Tyr Phe Gln His Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser 115
120230119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 230Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Pro Phe Ile Gly Gln 20 25 30Tyr Leu His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Gly
Asp Ser Ala Thr Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Leu Ser Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly 100 105 110Thr
Thr Val Thr Val Ser Ser 115231122PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 231Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Met Asn Pro Ile Gly Gly Gly Thr Gly Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Tyr Asp Phe Trp Ser Val Leu Ser Gly Phe Asp Ile
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120232124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 232Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Asn Trp Asn Gly
Gly Ser Thr Gly Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val
Glu Gln Gly Tyr Asp Ile Tyr Tyr Tyr Tyr Tyr Met Asp 100 105 110Val
Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser 115
120233123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 233Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Arg Gly
Asp Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala
Ser Gly Ser Gly Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val 100 105 110Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120234118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
234Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Gly Asn
Tyr 20 25 30Phe Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Met Val Asn Pro Ser Gly Gly Ser Glu Thr Phe Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ser Thr Trp Ile Gln Pro Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
115235122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 235Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Asp Phe Ser Ile Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Asn
Gly Asn Tyr Tyr Gly Ser Gly Ser Tyr Tyr Asn Tyr Trp 100 105 110Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120236122PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
236Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Thr
Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Val Tyr Tyr Asp Phe Trp
Ser Gly Pro Phe Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120237121PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 237Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Pro Tyr Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Gly Gly Ile Tyr Tyr Gly Ser Gly Ser Tyr Pro Ser Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
120238116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 238Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Ser Pro Tyr Ser
Gly Asn Thr Asp Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Leu Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val 100 105 110Thr
Val Ser Ser 115239123PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 239Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn Met 20 25 30Tyr Leu His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asn Pro Asn Thr Gly Asp Thr Asn Tyr Ala Gln Thr Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Leu Tyr Gly Asp Tyr Phe Leu Tyr Tyr Gly Met Asp
Val 100 105 110Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser 115
120240124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 240Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Met Asn Pro Asn Ser
Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Leu Leu Gly Phe Gly Glu Phe Leu Thr Tyr Gly Met Asp 100 105 110Val
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120241124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 241Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Ile His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Val Ile Asn Pro Ser Gly
Gly Ser Thr Thr Tyr Ala Gln Lys Leu 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Arg Asp Ser Ser Trp Thr Tyr Tyr Tyr Tyr Gly Met Asp 100 105 110Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120242123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 242Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Asn 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Met Asn Pro Asn Ser
Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Leu Tyr Gly Asp Tyr Phe Leu Tyr Tyr Gly Met Asp Val 100 105 110Trp
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120243125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
243Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
His 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Val Ile Ile Pro Ser Gly Gly Thr Ser Tyr Thr Gln
Lys Phe Gln 50 55 60Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr Met65 70 75 80Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Asp Tyr Tyr Asp Ser Ser Gly
Tyr Tyr Phe Pro Val Tyr Phe 100 105 110Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 125244124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
244Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Pro Phe Trp Ser Gly His
Tyr Tyr Tyr Tyr Gly Met Asp 100 105 110Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120245106PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 245Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Thr Ser Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Tyr Asn Ser Val Thr
85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105246107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 246Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Trp Ala
Ser Gln Gly Ile Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Asn Thr Pro Trp 85 90 95Thr Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys 100 105247107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
247Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Ser Asn
Ser 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln
Ser Tyr Ser Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105248107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 248Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys
Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Tyr
85 90
95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100
105249107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 249Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Tyr 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 105250107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
250Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
His Asn Ser Tyr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105251107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 251Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Tyr Pro Ile
85 90 95Thr Ile Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105252107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 252Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Asn Ser 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Trp 85 90 95Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105253107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
253Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser His Ser Thr Pro Gln 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105254107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 254Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105255107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 255Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 105256107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
256Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn
Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Thr Tyr Ser Thr Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105257107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 257Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Asn Ser 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Gly
Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105258107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 258Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Gly Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser His Ser Thr Pro Leu 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105259107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
259Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Tyr Thr
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ala Asn Gly Phe Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 10526010PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 260Ala Ser Ser Leu Pro Thr Thr Met Asn
Tyr1 5 1026119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 261Cys Ala Arg Asp Gln Asp Thr Ile Phe
Gly Val Val Ile Thr Trp Phe1 5 10 15Asp Pro Trp26214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 262Cys
Ala Arg Asp Lys Val Tyr Gly Asp Gly Phe Asp Pro Trp1 5
1026311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 263Cys Ala Arg Glu Asp Asp Ser Met Asp Val Trp1 5
1026411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 264Cys Ala Arg Asp Ser Ser Gly Leu Asp Pro Trp1 5
1026511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 265Cys Ala Arg Gly Val Gly Asn Leu Asp Tyr Trp1 5
1026627PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 266Cys Ala Arg Asp Ala His Gln Tyr Tyr Asp Phe
Trp Ser Gly Tyr Tyr1 5 10 15Ser Gly Thr Tyr Tyr Tyr Gly Met Asp Val
Trp 20 2526715PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 267Cys Ala Arg Glu Gln Trp Pro Ser Tyr
Trp Tyr Phe Asp Leu Trp1 5 10 1526815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 268Cys
Ala Arg Asp Arg Gly Tyr Ser Tyr Gly Tyr Phe Asp Tyr Trp1 5 10
1526919PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 269Cys Ala Arg Gly Ser Gly Asp Pro Asn Tyr Tyr
Tyr Tyr Tyr Gly Leu1 5 10 15Asp Val Trp27012PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 270Cys
Ala Arg Asp Thr Gly Asp His Phe Asp Tyr Trp1 5 1027111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 271Cys
Ala Arg Ala Glu Asn Gly Met Asp Val Trp1 5 1027212PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 272Cys
Ala Arg Asp Pro Gly Gly Tyr Met Asp Val Trp1 5 1027311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 273Cys
Ala Arg Asp Gly Asp Ala Phe Asp Ile Trp1 5 1027412PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 274Cys
Ala Arg Asp Met Gly Asp Ala Phe Asp Ile Trp1 5 1027511PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 275Cys
Ala Arg Glu Glu Asp Gly Met Asp Val Trp1 5 1027619PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 276Cys
Ala Arg Gly Glu Tyr Ser Ser Gly Phe Phe Phe Val Gly Trp Phe1 5 10
15Asp Leu Trp27713PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 277Cys Ala Arg Glu Thr Gly Asp Asp Ala
Phe Asp Ile Trp1 5 1027811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 278Cys Gln Gln Tyr Phe Thr
Thr Pro Tyr Thr Phe1 5 1027911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 279Cys Gln Gln Ala Glu Ala
Phe Pro Tyr Thr Phe1 5 1028011PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 280Cys Gln Gln Ser Tyr Ser
Thr Pro Ile Thr Phe1 5 1028111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 281Cys Gln Gln Ser Tyr Ile
Ile Pro Tyr Thr Phe1 5 1028211PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 282Cys His Gln Thr Tyr Ser
Thr Pro Leu Thr Phe1 5 1028311PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 283Cys Gln Gln Ala Tyr Ser
Phe Pro Trp Thr Phe1 5 1028411PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 284Cys Gln Gln Gly Tyr Ser
Thr Pro Leu Thr Phe1 5 1028511PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 285Cys Gln Gln Ala Asn Ser
Phe Pro Arg Thr Phe1 5 1028611PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 286Cys Gln Gln Ala Asn Ser
Leu Pro Tyr Thr Phe1 5 1028710PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 287Cys Gln Gln Ser Tyr Gly
Val Pro Thr Phe1 5 1028811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 288Cys Gln Gln Tyr Tyr Ser
Tyr Pro Trp Thr Phe1 5 1028911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 289Cys Met Gln Thr Leu Lys
Thr Pro Leu Ser Phe1 5 10290124PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 290Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly
Ile Ser Ala Arg Ser Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Gln Asp Thr Ile Phe Gly Val Val Ile Thr Trp Phe
Asp 100 105 110Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120291119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 291Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile His Pro Gly Gly
Gly Thr Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Lys Val Tyr Gly Asp Gly Phe Asp Pro Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 115292116PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 292Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Gly Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Met
Ile Gly Pro Ser Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Asp Asp Ser Met Asp Val Trp Gly Lys Gly Thr Thr
Val 100 105 110Thr Val Ser Ser 115293116PRTArtificial
SequenceDescription of Artificial
Sequence Synthetic polypeptide 293Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Ile Gly Tyr 20 25 30Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Met Ile Gly Pro
Ser Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asp Ser Ser Gly Leu Asp Pro Trp Gly Gln Gly Thr Leu Val 100 105
110Thr Val Ser Ser 115294116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 294Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Met
Ile Gly Pro Ser Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Val Gly Asn Leu Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser 115295132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
295Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Val Thr Phe Ser Thr
Ser 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Ser Pro Tyr Asn Gly Asn Thr Asp Tyr Ala
Gln Met Leu 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Ala His Gln Tyr Tyr Asp
Phe Trp Ser Gly Tyr Tyr Ser 100 105 110Gly Thr Tyr Tyr Tyr Gly Met
Asp Val Trp Gly Gln Gly Thr Thr Val 115 120 125Thr Val Ser Ser
130296120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 296Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Asn Ser 20 25 30Ile Ile Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Met Asn Pro Asn Ser
Gly Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu
Gln Trp Pro Ser Tyr Trp Tyr Phe Asp Leu Trp Gly Arg 100 105 110Gly
Thr Leu Val Thr Val Ser Ser 115 120297120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
297Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr
His 20 25 30Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Val Ile Asn Pro Ser Gly Gly Ser Ala Ile Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Arg Gly Tyr Ser Tyr Gly
Tyr Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser 115 120298124PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 298Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Asn Thr Phe Ile Gly Tyr 20 25 30Tyr Val His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45Gly Ile Ile Asn Pro
Asn Gly Gly Ser Ile Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gly Ser Gly Asp Pro Asn Tyr Tyr Tyr Tyr Tyr Gly Leu Asp 100 105
110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120299117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 299Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Leu Ser Tyr Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Met Ile Gly Pro Ser Asp
Gly Ser Thr Ser Tyr Ala Gln Arg Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Gly Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Thr Gly Asp His Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 115300116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 300Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Gly Pro Ser Asp Gly Ser Thr Thr Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ala Glu Asn Gly Met Asp Val Trp Gly Gln Gly Thr Thr
Val 100 105 110Thr Val Ser Ser 115301117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
301Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly
Tyr 20 25 30Tyr Val His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ile Ile Ala Pro Ser Asp Gly Ser Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Pro Gly Gly Tyr Met Asp
Val Trp Gly Lys Gly Thr Thr 100 105 110Val Thr Val Ser Ser
115302116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 302Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Leu His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Met Ile Gly Pro Ser Asp
Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110Thr
Val Ser Ser 115303117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 303Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg
Ile Ser Pro Ser Asp Gly Ser Thr Thr Tyr Ala Pro Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Met Gly Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
Thr 100 105 110Val Thr Val Ser Ser 115304116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
304Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly
Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Met Ile Gly Pro Ser Asp Gly Ser Thr Ser Tyr Ala
Gln Arg Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Glu Asp Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val 100 105 110Thr Val Ser Ser
115305124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 305Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Asn Asn Phe 20 25 30Ala Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe
Asp Ala Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Phe
Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Glu Tyr Ser Ser Gly Phe Phe Phe Val Gly Trp Phe Asp 100 105 110Leu
Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 115
120306118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 306Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Asn Phe Thr Gly Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Ala Pro Ser Asp
Gly Ser Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu
Thr Gly Asp Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110Met
Val Thr Val Ser Ser 115307107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 307Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Thr Thr Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105308107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 308Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Phe Asp Ala Ser Arg Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ala Glu Ala Phe Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105309107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
309Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Thr Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys 100 105310107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 310Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys
Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ile Ile Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105311107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 311Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys His Gln Thr Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105312107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
312Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40
45Tyr Ser Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser
Phe Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105313107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 313Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asn Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys 100 105314107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
314Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Arg
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ala Asn Ser Phe Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105315107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 315Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Leu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105316107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 316Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Thr Leu Gln
Asn Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe 85 90 95Thr Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys 100 105317107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
317Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg Ile Ser Ser
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Thr Pro Phe 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys 100 105318106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 318Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Lys Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Gly Val Pro Thr
85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105319107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 319Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Leu Glu
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 105320107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
320Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Thr
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Tyr Ser Tyr Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu
Ile Lys 100 105321112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 321Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr
Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln
Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Thr
85 90 95Leu Lys Thr Pro Leu Ser Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105 1103229PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 322Leu Leu Ala Ser Ser Ile Leu Cys Ala1
532318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 323Cys Ala Arg Asp Gly Tyr Asp Phe Trp Ser Gly
Tyr Thr Ser Asp Asp1 5 10 15Tyr Trp3249PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 324Cys
Ala Ser Asp Tyr Gly Asp Tyr Arg1 532520PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 325Cys
Ala Arg Asp Leu Met Thr Thr Val Val Thr Pro Gly Asp Tyr Gly1 5 10
15Met Asp Val Trp 2032614PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 326Cys Ala Arg Gln Asp Gly
Gly Ala Phe Ala Phe Asp Ile Trp1 5 1032714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 327Cys
Ala Arg Glu Leu Gly Tyr Tyr Tyr Gly Met Asp Val Trp1 5
1032819PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 328Cys Ala Arg Ala Leu Ile Phe Gly Val Pro Leu
Leu Pro Tyr Gly Met1 5 10 15Asp Val Trp32920PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 329Cys
Ala Lys Asp Leu Ala Thr Val Gly Glu Pro Tyr Tyr Tyr Tyr Gly1 5 10
15Met Asp Val Trp 2033020PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 330Cys Ala Arg Leu Trp Phe
Gly Glu Leu His Tyr Tyr Tyr Tyr Tyr Gly1 5 10 15Met Asp Val Trp
2033110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 331Cys His His Tyr Gly Arg Ser His Thr Phe1 5
1033211PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 332Cys Gln Gln Ala Asn Ala Phe Pro Pro Thr Phe1 5
1033311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 333Cys Gln Gln Tyr Tyr Ser Ile Pro Leu Thr Phe1 5
1033411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 334Cys Gln Gln Ser Tyr Ser Thr Pro Pro Thr Phe1 5
1033511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 335Cys Gln Gln Ser Tyr Ser Phe Pro Tyr Thr Phe1 5
1033611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 336Cys Gln Gln Gly Asn Thr Phe Pro Leu Thr Phe1 5
1033711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 337Cys Met Gln Gly Ser His Trp Pro Pro Ser Phe1 5
10338122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 338Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Asn Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Gly Gly
Ser Thr Ser Tyr Ala Gln Lys Phe Gln 50 55 60Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Val Tyr Met65 70 75 80Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Asp Gly
Tyr Asp Phe Trp Ser Gly Tyr Thr Ser Asp Asp Tyr Trp 100 105 110Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120339114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
339Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Gly Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Asp Tyr Gly Asp Tyr Arg Gly
Gln Gly Thr Leu Val Thr Val 100 105 110Ser Ser340125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
340Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asn
Tyr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Leu Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala
Gln Arg Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Met Thr Thr Val Val
Thr Pro Gly Asp Tyr Gly Met 100 105 110Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120 125341119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
341Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Met Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr
Asp 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Arg Ile Tyr Pro His Ser Gly Tyr Thr Glu Tyr Ala
Lys Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gln Asp Gly Gly Ala Phe Ala
Phe Asp Ile Trp Gly Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser
115342119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 342Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Gln 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Ser Pro Asn Asn
Gly Asp Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu
Leu Gly Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly 100 105 110Thr
Thr Val Thr Val Ser Ser 115343124PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 343Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Arg Tyr Thr Phe Thr Ser Tyr 20 25 30Asp Ile Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg
Ile Ile Pro Met Leu Asn Ile Ala Asn Tyr Ala Pro Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ala Leu Ile Phe Gly Val Pro Leu Leu Pro Tyr Gly Met
Asp 100 105 110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120344125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 344Glu Val Gln Leu Leu Gln Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Ser 20 25 30Trp Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Phe Ile Ser Thr Ser Ser
Gly Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp
Leu Ala Thr Val Gly Glu Pro Tyr Tyr Tyr Tyr Gly Met 100 105 110Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
125345125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 345Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Asp Thr Phe Asn Thr Tyr 20 25 30Ala Leu Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Met Asn Pro Asn Ser
Gly Asn Ala Gly Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu
Trp Phe Gly Glu Leu His Tyr Tyr Tyr Tyr Tyr Gly Met 100 105 110Asp
Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
125346107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 346Glu Ile Val Met Thr Gln Ser Pro Ala Thr
Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Ser 20 25 30Asn Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Thr Arg
Ala Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Ser Glu Asp Phe
Ala Val Tyr Tyr Cys His His Tyr Gly Arg Ser His 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105347107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
347Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn
Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ala Asn Ala Phe Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105348113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 348Asp Ile Val Met Thr
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr
Ile Asn Cys Lys Ser Ser Gln Ser Val Phe Tyr Ser 20 25 30Ser Asn Asn
Lys Asn Gln Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75
80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95Tyr Tyr Ser Ile Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile 100 105 110Lys349107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 349Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Asp Ile Phe Lys Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
105350107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 350Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Thr Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Phe Pro Tyr 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105351112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
351Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Ser Ser Ser Gln Ser Leu Leu His
Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr Pro Leu Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 110352107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
352Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Asn Leu Arg Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Phe Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105353112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 353Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr
Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln
Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly
85 90 95Ser His Trp Pro Pro Ser Phe Gly Gln Gly Thr Arg Leu Glu Ile
Lys 100 105 1103549PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 354Glu Val Asp Pro Ile Gly His Leu Tyr1
53559PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 355Gly Val His Gly Gly Ile Leu Asn Lys1
535610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 356Gly Val Tyr Asp Gly Glu Glu His Ser Val1 5
1035710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 357Gly Glu Met Ser Ser Asn Ser Thr Ala Leu1 5
103589PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 358Ile Pro Ser Ile Asn Val His His Tyr1
53599PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 359Phe Leu Leu Thr Arg Ile Leu Thr Ile1
53609PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 360Ala Thr Asp Ala Leu Met Thr Gly Tyr1
53615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 361Gly Gly Gly Gly Ser1 53624PRTHomo sapiens
362Glu Leu Leu Gly13634PRTHomo sapiens 363Glu Phe Leu
Gly136422PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 364Ala Ala Asp Met Ala Ala Gln Thr Thr Lys His
Lys Trp Glu Ala Ala1 5 10 15His Val Ala Glu Gln Leu
20365117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 365Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Ala 20 25 30Thr Ile Ser Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Tyr Pro Asn Ser
Gly Gly Thr Val Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Thr
Glu Trp Leu Gly Val Trp Gly Gln Gly Thr Thr Val Thr 100 105 110Val
Ser Ser Ala Ser 11536615PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 366Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 153679PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 367His
Ser Glu Val Gly Leu Pro Val Tyr1 53689PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 368Leu
Leu Phe Gly Tyr Pro Val Tyr Val1 53699PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 369Gly
Ile Leu Gly Phe Val Phe Thr Leu1 537011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 370Tyr
Ser Glu His Pro Thr Phe Thr Ser Gln Tyr1 5 103719PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 371Val
Ser Asp Gly Gly Pro Asn Leu Tyr1 53729PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 372Ile
Val Thr Asp Phe Ser Val Ile Lys1 53739PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 373Lys
Ser Met Arg Glu Glu Tyr Arg Lys1 537410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 374Ser
Ser Cys Ser Ser Cys Pro Leu Ser Lys1 5 103759PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 375Ala
Thr Ile Gly Thr Ala Met Tyr Lys1 537610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 376Ala
Val Phe Asp Arg Lys Ser Asp Ala Lys1 5 103779PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 377Ser
Ile Ile Pro Ser Gly Pro Leu Lys1 537811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 378Glu
Pro Leu Pro Gln Gly Gln Leu Thr Ala Tyr1 5 1037910PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 379Val
Pro Leu Asp Glu Asp Phe Arg Lys Tyr1 5 103809PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 380Arg
Leu Arg Ala Glu Ala Gln Val Lys1 53819PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 381Arg
Leu Arg Pro Gly Gly Lys Lys Lys1 538210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 382Gln
Val Pro Leu Arg Pro Met Thr Tyr Lys1 5 103839PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 383Thr
Tyr Gly Pro Val Phe Met Cys Leu1 53849PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 384Arg
Tyr Leu Lys Asp Gln Gln Leu Leu1 53859PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 385Pro
Tyr Leu Phe Trp Leu Ala Ala Ile1 5386121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
386Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ile Ile Asn Pro Arg Ser Gly Ser Thr Lys Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Val Arg Tyr Tyr Gly
Met Asp Val Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
Ala Ser 115 1203879PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 387Tyr Thr Phe Thr Ser Tyr Asp Ile Asn1
538813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 388Gly Ile Ile Asn Pro Arg Ser Gly Ser Thr Lys
Tyr Ala1 5 1038916PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 389Arg Ser Ser Gln Ser Leu Leu His Ser
Asn Gly Tyr Asn Tyr Leu Asp1 5 10 153907PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 390Leu
Gly Ser Tyr Arg Ala Ser1 539112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 391Cys Ala Gly Pro Gly Asn
Thr Gly Lys Leu Ile Phe1 5 1039213PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 392Cys Ala Ser Ser Asn Ala
Gly Asp Gln Pro Gln His Phe1 5 10393127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
393Met Leu Leu Ile Thr Ser Met Leu Val Leu Trp Met Gln Leu Ser Gln1
5 10 15Val Asn Gly Gln Gln Val Met Gln Ile Pro Gln Tyr Gln His Val
Gln 20 25 30Glu Gly Glu Asp Phe Thr Thr Tyr Cys Asn Ser Ser Thr Thr
Leu Ser 35 40 45Asn Ile Gln Trp Tyr Lys Gln Arg Pro Gly Gly His Pro
Val Phe Leu 50 55 60Ile Gln Leu Val Lys Ser Gly Glu Val Lys Lys Gln
Lys Arg Leu Thr65 70 75 80Phe Gln Phe Gly Glu Ala Lys Lys Asn Ser
Ser Leu His Ile Thr Ala 85 90 95Thr Gln Thr Thr Asp Val Gly Thr Tyr
Phe Cys Ala Gly Pro Gly Asn 100 105 110Thr Gly Lys Leu Ile Phe Gly
Gln Gly Thr Thr Leu Gln Val Lys 115 120 125394131PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
394Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu Gly Ala1
5 10 15Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe
Arg 20 25 30Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu
Asn His 35 40 45Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly
Leu Arg Leu 50 55 60Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys
Gly Asp Ile Ala65 70 75 80Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys
Glu Ser Phe Pro Leu Thr 85 90 95Val Thr Ser Ala Gln Lys Asn Pro Thr
Ala Phe Tyr Leu Cys Ala Ser 100 105 110Ser Asn Ala Gly Asp Gln Pro
Gln His Phe Gly Asp Gly Thr Arg Leu 115 120 125Ser Ile Leu
1303959PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 395Phe Val Gln Glu Asn Tyr Leu Glu Tyr1
53969PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 396Leu Val His Phe Leu Leu Leu Lys Tyr1
539710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 397Met Glu Val Asp Pro Ile Gly
His Leu Tyr1 5 103989PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 398Glu Val Asp Pro Ala Ser
Asn Thr Tyr1 53999PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 399Lys Val Leu Glu Tyr Val Ile Lys Val1
54009PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 400Leu Val Gln Glu Asn Tyr Leu Glu Tyr1
54019PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 401Ala Phe Asp Asp Ile Ala Thr Tyr Phe1
54029PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 402Trp Val Gln Glu Asn Tyr Leu Glu Tyr1
54039PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 403Ser Leu Phe Arg Ala Val Ile Thr Lys1
54049PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 404Ala Leu Ala Glu Thr Ser Tyr Val Lys1
54059PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 405Leu Tyr Ala Thr Val Ile His Asp Ile1
540610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 406Gln Leu Leu Asp Gly Phe Met Ile Thr Leu1 5
104079PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 407Ser Thr Leu Pro Thr Thr Ile Asn Tyr1
540810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 408Tyr Pro Ala Pro Leu Glu Ser Leu Asp Tyr1 5
104099PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 409Ala Thr Leu Glu Asn Leu Leu Ser His1
54108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 410Asp Ala Leu Leu Ala Gln Lys Val1
541110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 411Ser Glu Ser Asp Leu Lys His Leu Ser Trp1 5
104129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 412Thr Leu Asp Glu Tyr Leu Thr Tyr Leu1
54139PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 413Arg Gln Lys Arg Ile Leu Val Asn Leu1
54149PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 414Leu Val Ile Asp Thr Val Thr Glu Val1
541510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 415Glu Val Asp Pro His Ile Gly His Leu Tyr1 5
104169PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 416Tyr Thr Phe Thr Ser His Asp Ile Asn1
54179PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 417Phe Ser Phe Ser Ser Tyr Trp Met Ser1
54189PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 418Phe Thr Phe Ser Asn Ser Asp Met Asn1
54199PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 419Gly Thr Phe Ser Asn Phe Gly Val Ser1
54209PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 420Tyr Thr Phe Thr Ser Tyr Asn Met His1
54219PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 421Gly Thr Phe Ser Gly Tyr Leu Val Ser1
54229PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 422Tyr Ile Phe Arg Asn Tyr Pro Met His1
54239PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 423Tyr Thr Phe Thr Gly Tyr Tyr Met His1
54249PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 424Gly Thr Phe Ser Ser Tyr Gly Ile Ser1
54259PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 425Phe Thr Phe Ser Asp Tyr Tyr Met Ser1
54269PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 426Gly Thr Phe Ser Ser Tyr Ala Ile Ser1
54279PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 427Phe Thr Phe Thr Ser Tyr Ser Met His1
54289PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 428Tyr Thr Phe Thr Asn Tyr Tyr Met His1
54299PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 429Tyr Thr Phe Thr Ser Tyr Tyr Met His1
543013PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 430Gly Trp Met Asn Pro Asn Ser Gly Asp Thr Gly
Tyr Ala1 5 1043113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 431Ser Tyr Ile Ser Gly Asp Ser Gly Tyr
Thr Asn Tyr Ala1 5 1043213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 432Ala Tyr Ile Ser Ser Gly
Ser Ser Thr Ile Tyr Tyr Ala1 5 1043313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 433Ala
Ser Ile Ser Ser Ser Gly Gly Tyr Ile Asn Tyr Ala1 5
1043413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 434Gly Gly Ile Ile Pro Ile Leu Gly Thr Ala Asn
Tyr Ala1 5 1043513PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 435Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr Asn Tyr Ala1 5 1043613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 436Gly Trp Ile Asn Pro Asn
Ser Gly Gly Thr Asn Thr Ala1 5 1043713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 437Gly
Trp Ile Asn Pro Asp Ser Gly Gly Thr Lys Tyr Ala1 5
1043813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 438Gly Trp Met Asn Pro Asn Ile Gly Asn Thr Gly
Tyr Ala1 5 1043913PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 439Gly Trp Ile Asn Pro Asn Ser Gly Val
Thr Lys Tyr Ala1 5 1044013PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 440Gly Trp Ile Asn Pro Asn
Ser Gly Asp Thr Lys Tyr Ser1 5 1044113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 441Ser
Tyr Ile Ser Ser Ser Ser Ser Tyr Thr Asn Tyr Ala1 5
1044213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 442Gly Trp Met Asn Pro Asp Ser Gly Ser Thr Gly
Tyr Ala1 5 1044313PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 443Ser Ser Ile Thr Ser Phe Thr Asn Thr
Met Tyr Tyr Ala1 5 1044413PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 444Gly Ile Ile Asn Pro Ser
Gly Gly Ser Thr Ser Tyr Ala1 5 1044513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 445Gly
Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala1 5
1044613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 446Gly Gly Ile Ile Pro Val Met Gly Thr Gly Asn
Tyr Ala1 5 1044711PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 447Gln Ala Ser Gln Asp Ile Ser Asn Tyr
Leu Asn1 5 1044811PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 448Arg Ala Ser Gln Ser Ile Ser Ser Trp
Leu Ala1 5 1044911PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 449Arg Ala Ser Gln Gly Ile Ser Asn Tyr
Leu Asn1 5 1045011PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 450Arg Ala Ser Gln Ser Ile Ser Ser Tyr
Leu Asn1 5 1045111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 451Arg Ala Ser Gln Gly Ile Ser Asn Tyr
Leu Ala1 5 1045217PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 452Lys Thr Ser Gln Ser Val Leu Tyr Arg
Pro Asn Asn Glu Asn Tyr Leu1 5 10 15Ala45311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 453Arg
Ala Ser Gln Ser Ile Ser Arg Phe Leu Asn1 5 1045411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 454Arg
Ala Ser Gln Ser Val Ser Ser Asn Leu Ala1 5 1045511PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 455Gln
Ala Ser Glu Asp Ile Ser Asn His Leu Asn1 5 104567PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 456Leu
Gly Ser Ser Arg Ala Ser1 54577PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 457Ala Ala Ser Ser Leu Gln
Ser1 54587PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 458Ser Ala Ser Thr Leu Gln Ser1
54597PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 459Ala Ala Ser Thr Leu Gln Ser1
54607PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 460Tyr Ala Ser Ser Leu Gln Ser1
54617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 461Gly Ala Ser Ser Leu Gln Ser1
54627PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 462Gln Ala Ser Ile Arg Glu Pro1
54637PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 463Gly Ala Ser Arg Pro Gln Ser1
54647PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 464Leu Gly Ser His Arg Ala Ser1
54657PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 465Ala Ala Ser Ala Arg Ala Ser1
54667PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 466Gly Ala Ser Arg Leu Gln Ser1
54677PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 467Leu Gly Ser Asn Arg Ala Ser1
54687PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 468Asp Ala Leu Ser Leu Gln Ser1
54699PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 469Gly Thr Phe Ser Arg Ser Ala Ile Thr1
54709PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 470Tyr Pro Phe Ile Gly Gln Tyr Leu His1
54719PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 471Gly Thr Leu Ser Ser Tyr Pro Ile Asn1
54729PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 472Phe Thr Phe Ser Ser Tyr Trp Met Ser1
54739PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 473Tyr Thr Phe Gly Asn Tyr Phe Met His1
54749PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 474Phe Asp Phe Ser Ile Tyr Ser Met Asn1
54759PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 475Tyr Thr Leu Thr Thr Tyr Tyr Met His1
54769PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 476Gly Thr Phe Ser Ser Tyr Gly Val Ser1
54779PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 477Tyr Thr Phe Ser Asn Met Tyr Leu His1
54789PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 478Tyr Thr Phe Thr Gly Tyr Tyr Ile His1
54799PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 479Tyr Thr Phe Thr Ser Asn Tyr Met His1
54809PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 480Gly Thr Phe Ser Ser His Ala Ile Ser1
54819PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 481Tyr Thr Phe Thr Ser Tyr Ala Met Asn1
548213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 482Gly Trp Ile Asn Pro Asn Ser Gly Ala Thr Asn
Tyr Ala1 5 1048313PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 483Gly Ile Ile Asn Pro Ser Gly Asp Ser
Ala Thr Tyr Ala1 5 1048413PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 484Gly Trp Met Asn Pro Ile
Gly Gly Gly Thr Gly Tyr Ala1 5 1048513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 485Ser
Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala1 5
1048613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 486Gly Trp Ile Ser Thr Tyr Ser Gly His Ala Asp
Tyr Ala1 5 1048713PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 487Ser Ser Ile Ser Gly Arg Gly Asp Asn
Thr Tyr Tyr Ala1 5 1048813PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 488Gly Met Val Asn Pro Ser
Gly Gly Ser Glu Thr Phe Ala1 5 1048913PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 489Ser
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala1 5
1049013PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 490Gly Trp Ile Asn Pro Tyr Ser Gly Gly Thr Asn
Tyr Ala1 5 1049113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 491Gly Trp Ile Ser Pro Tyr Ser Gly Asn
Thr Asp Tyr Ala1 5 1049213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 492Gly Trp Ile Asn Pro Asn
Thr Gly Asp Thr Asn Tyr Ala1 5 1049313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 493Gly
Val Ile Asn Pro Ser Gly Gly Ser Thr Thr Tyr Ala1 5
1049412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 494Gly Val Ile Ile Pro Ser Gly Gly Thr Ser Tyr
Thr1 5 1049511PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 495Arg Ala Ser Gln Ser Ile Thr Ser Tyr
Leu Asn1 5 1049611PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 496Trp Ala Ser Gln Gly Ile Ser Ser Tyr
Leu Ala1 5 1049711PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 497Arg Ala Ser Gln Ala Ile Ser Asn Ser
Leu Ala1 5 1049811PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 498Arg Ala Ser Gln Gly Ile Asn Ser Tyr
Leu Ala1 5 1049911PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 499Arg Ala Ser Gln Ser Ile Ser Arg Trp
Leu Ala1 5 1050011PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 500Arg Ala Ser Gln Gly Ile Ser Asn Ser
Leu Ala1 5 1050111PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 501Arg Ala Ser Gln Asp Val Ser Thr Trp
Leu Ala1 5 1050211PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 502Arg Ala Ser Gln Gly Ile Ser Asn Trp
Leu Ala1 5 1050311PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 503Arg Ala Ser Gln Ser Val Gly Asn Ser
Leu Ala1 5 1050411PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 504Arg Ala Ser Gln Ser Ile Ser Gly Tyr
Leu Asn1 5 1050511PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 505Arg Ala Ser Gln Asn Ile Tyr Thr Tyr
Leu Asn1 5 105067PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 506Asp Ala Ser Asn Leu Glu Thr1
55077PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 507Lys Ala Ser Ser Leu Glu Ser1
55087PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 508Gly Ala Ser Thr Arg Ala Thr1
55099PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 509Tyr Ile Phe Thr Gly Tyr Tyr Met His1
55109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 510Tyr Thr Phe Ile Gly Tyr Tyr Met His1
55119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 511Val
Thr Phe Ser Thr Ser Ala Ile Ser1 55129PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 512Gly
Thr Phe Ser Asn Ser Ile Ile Asn1 55139PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 513Gly
Thr Phe Ser Thr His Asp Ile Asn1 55149PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 514Asn
Thr Phe Ile Gly Tyr Tyr Val His1 55159PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 515Tyr
Thr Leu Ser Tyr Tyr Tyr Met His1 55169PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 516Tyr
Thr Phe Thr Gly Tyr Tyr Val His1 55179PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 517Tyr
Thr Phe Thr Gly Tyr Tyr Leu His1 55189PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 518Gly
Thr Phe Asn Asn Phe Ala Ile Ser1 55199PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 519Tyr
Asn Phe Thr Gly Tyr Tyr Met His1 552013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 520Ser
Gly Ile Ser Ala Arg Ser Gly Arg Thr Tyr Tyr Ala1 5
1052113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 521Gly Ile Ile His Pro Gly Gly Gly Thr Thr Ser
Tyr Ala1 5 1052213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 522Gly Met Ile Gly Pro Ser Asp Gly Ser
Thr Ser Tyr Ala1 5 1052313PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 523Gly Trp Ile Ser Pro Tyr
Asn Gly Asn Thr Asp Tyr Ala1 5 1052413PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 524Gly
Trp Met Asn Pro Asn Ser Gly Asn Thr Asn Tyr Ala1 5
1052513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 525Gly Val Ile Asn Pro Ser Gly Gly Ser Ala Ile
Tyr Ala1 5 1052613PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 526Gly Ile Ile Asn Pro Asn Gly Gly Ser
Ile Ser Tyr Ala1 5 1052713PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 527Gly Ile Ile Gly Pro Ser
Asp Gly Ser Thr Thr Tyr Ala1 5 1052813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 528Gly
Ile Ile Ala Pro Ser Asp Gly Ser Thr Asn Tyr Ala1 5
1052913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 529Gly Arg Ile Ser Pro Ser Asp Gly Ser Thr Thr
Tyr Ala1 5 1053013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 530Gly Gly Ile Ile Pro Ile Phe Asp Ala
Thr Asn Tyr Ala1 5 1053111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 531Arg Ala Ser Gln Ser Ile
Ser Asn Tyr Leu Asn1 5 1053211PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 532Arg Ala Ser Gln Asn Ile
Ser Ser Tyr Leu Asn1 5 1053311PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 533Arg Ala Ser Gln Asp Ile
Ser Arg Tyr Leu Ala1 5 1053411PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 534Arg Ala Ser Gln Arg Ile
Ser Ser Tyr Leu Asn1 5 1053511PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 535Arg Ala Ser Gln Ser Ile
Ser Ser Tyr Leu Ala1 5 1053611PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 536Arg Ala Ser Gln Gly Ile
Ser Ser Trp Leu Ala1 5 1053711PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 537Arg Ala Ser Gln Gly Ile
Ser Thr Tyr Leu Ala1 5 105387PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 538Ala Ala Ser Ser Leu Gln
Gly1 55397PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 539Asp Ala Ser Arg Leu Gln Ser1
55407PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 540Ser Ala Ser Asn Leu Gln Ser1
55417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 541Ala Ala Ser Asn Leu Gln Ser1
55427PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 542Ala Ala Ser Thr Leu Gln Asn1
55437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 543Asp Ala Ser Lys Leu Glu Thr1
55447PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 544Asp Ala Ser Ser Leu Gln Ser1
55459PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 545Gly Thr Phe Ser Ser Ala Thr Ile Ser1
55469PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 546Tyr Thr Phe Thr Thr Tyr Asp Leu Ala1
55479PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 547Tyr Ser Phe Asp Ser Tyr Val Val Asn1
55489PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 548Tyr Thr Phe Thr Ser Tyr Gly Ile Ser1
55499PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 549Tyr Thr Phe Thr Arg Tyr Thr Ile Asn1
55509PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 550Tyr Thr Phe Thr Ser Tyr Gly Ile Thr1
55519PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 551Gly Thr Phe Ser Asn Tyr Ile Leu Ser1
55529PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 552Tyr Ser Phe Thr Arg Tyr Asn Met His1
55539PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 553Gly Thr Phe Asn Asn Tyr Ala Phe Ser1
55549PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 554Tyr Thr Phe Ser Ser Tyr Asn Met His1
55559PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 555Gly Thr Phe Ser Ser Tyr Ala Phe Ser1
55569PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 556Tyr Thr Phe Thr Asp Tyr Asn Met His1
55579PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 557Tyr Thr Phe Thr Ser Tyr Leu Met His1
55589PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 558Tyr Thr Phe Ser Asp Tyr Tyr Val His1
55599PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 559Tyr Thr Phe Thr Thr Tyr Tyr Met His1
55609PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 560Gly Thr Phe Ser Asn Tyr Ala Ile Asn1
55619PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 561Tyr Thr Phe Thr Asp Tyr Tyr Met His1
55629PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 562Tyr Thr Leu Thr Ser His Leu Ile His1
55639PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 563Tyr Ser Phe Thr Asp Tyr Ile Val His1
55649PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 564Tyr Thr Phe Ser Asn Phe Leu Ile Asn1
55659PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 565Tyr Thr Phe Thr Asp Tyr Gln Met Phe1
55669PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 566Tyr Thr Phe Thr Asn Tyr His Met His1
55679PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 567Tyr Thr Phe Thr Ser Tyr Thr Val Asn1
55689PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 568Tyr Thr Phe Thr Ser Gln Tyr Met His1
556913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 569Gly Trp Ile Tyr Pro Asn Ser Gly Gly Thr Val
Tyr Ala1 5 1057013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 570Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr Ile Ser Ala1 5 1057113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 571Gly Trp Met Asn Pro Asn
Ser Gly Gly Thr Asn Tyr Ala1 5 1057213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 572Gly
Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr Ala1 5
1057313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 573Gly Trp Ile Asn Pro Asn Asn Gly Gly Thr Asn
Tyr Ala1 5 1057413PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 574Gly Trp Ile Asn Gly Asn Thr Gly Gly
Thr Asn Tyr Ala1 5 1057513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 575Gly Trp Ile Asn Pro Asp
Thr Gly Tyr Thr Arg Tyr Ala1 5 1057613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 576Gly
Trp Ile Ser Ala Tyr Asn Gly Tyr Thr Asn Tyr Ala1 5
1057713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 577Gly Trp Ile Asn Pro Asn Ser Gly Gly Ala Asn
Tyr Ala1 5 1057813PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 578Gly Trp Ile Ser Pro Asn Ser Gly Gly
Thr Asn Tyr Ala1 5 1057913PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 579Gly Trp Ile Ser Pro Tyr
Ser Gly Gly Thr Asn Tyr Ala1 5 1058013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 580Gly
Trp Ile Tyr Pro Asn Thr Gly Gly Thr Asn Tyr Ala1 5
1058113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 581Gly Trp Met Asn Pro Asn Ser Gly Gly Thr Lys
Tyr Ala1 5 1058213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 582Gly Trp Ile Asn Pro Tyr Ser Gly Gly
Thr Lys Tyr Ala1 5 1058313PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 583Gly Trp Ile His Pro Asp
Ser Gly Gly Thr Ser Tyr Ala1 5 1058413PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 584Gly
Trp Ile Asn Pro Asn Ser Gly Gly Thr Lys Tyr Ala1 5
1058513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 585Gly Met Ile Asn Pro Arg Asp Asp Thr Thr Asp
Tyr Ala1 5 1058613PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 586Gly Met Ile Asn Pro Ser Gly Gly Gly
Thr Ser Tyr Ala1 5 1058713PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 587Gly Arg Ile Ile Pro Leu
Leu Gly Ile Val Asn Tyr Ala1 5 1058811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 588Arg
Ala Ser Gln Ser Ile Ser Thr Trp Leu Ala1 5 1058911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 589Arg
Ala Ser Gln Asp Ile Ser Arg Trp Leu Ala1 5 1059011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 590Arg
Ala Ser Gln Thr Ile Ser Ser Trp Leu Ala1 5 1059111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 591Arg
Ala Ser Gln Ser Ile Ser Asn Trp Leu Ala1 5 1059211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 592Arg
Ala Ser Gln Ser Val Gly Asn Trp Leu Ala1 5 1059311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 593Arg
Ala Ser Gln Asn Ile Gly Asn Trp Leu Ala1 5 1059411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 594Arg
Ala Ser Gln Ser Ile Ser Lys Trp Leu Ala1 5 1059511PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 595Arg
Ala Ser Gln Thr Ile Ser Asn Tyr Leu Asn1 5 1059611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 596Arg
Ala Ser Arg Asp Ile Gly Arg Ala Val Gly1 5 1059711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 597Arg
Ala Ser Gln Ser Ile Gly Arg Trp Leu Ala1 5 1059811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 598Arg
Ala Ser Gln Ser Val Ser Asn Trp Leu Ala1 5 1059911PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 599Gln
Ala Ser Gln Asp Ile Gly Ser Trp Leu Ala1 5 1060011PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 600Arg
Ala Ser Gln Gly Ile Ser Arg Trp Leu Ala1 5 1060111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 601Arg
Ala Ser Gln Ser Ile Ser Ser Trp Val Ala1 5 1060211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 602Arg
Ala Ser Gln Ser Ile Ser His Tyr Leu Asn1 5 1060311PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 603Arg
Ala Ser Gln Ser Val Ser Arg Asn Leu Ala1 5 106047PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 604Ala
Ala Ser Ser Leu Arg Ser1 56057PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 605Ala Ala Ser Thr Val Gln
Ser1 56067PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 606Ala Ala Ser Arg Leu Gln Ala1
56077PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 607Gly Ala Ser Ser Leu Gln Thr1
56087PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 608Ala Ala Ser Thr Leu Gln Thr1
56097PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 609Ala Thr Ser Ser Leu Gln Ser1
56107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 610Ala Ala Ser Thr Leu Gln Pro1
56117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 611Ala Ala Ser Arg Leu Glu Ser1
56127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 612Gly Val Ser Ser Leu Gln Ser1
56137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 613Gly Ala Ser Asn Leu Glu Ser1
56149PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 614Gly Thr Phe Ser Asn Tyr Gly Ile Ser1
56159PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 615Phe Thr Phe Ser Ser Tyr Ala Met His1
56169PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 616Tyr Thr Phe Ser Asn Tyr Tyr Ile His1
56179PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 617Tyr Thr Phe Thr Thr Asp Gly Ile Ser1
56189PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 618Phe Thr Phe Ser Ser Ser Trp Met His1
56199PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 619Asp Thr Phe Asn Thr Tyr Ala Leu Ser1
562012PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 620Gly Ile Ile Asn Pro Gly Gly Ser Thr Ser Tyr
Ala1 5 1062113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 621Ser Gly Ile Ser Gly Ser Gly Gly Ser
Thr Tyr Tyr Ala1 5 1062213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 622Gly Trp Leu Asn Pro Asn
Ser Gly Asn Thr Gly Tyr Ala1 5 1062313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 623Gly
Arg Ile Tyr Pro His Ser Gly Tyr Thr Glu Tyr
Ala1 5 1062413PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 624Gly Trp Ile Ser Pro Asn Asn Gly Asp
Thr Asn Tyr Ala1 5 1062513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 625Gly Arg Ile Ile Pro Met
Leu Asn Ile Ala Asn Tyr Ala1 5 1062613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 626Ser
Phe Ile Ser Thr Ser Ser Gly Tyr Ile Tyr Tyr Ala1 5
1062713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 627Gly Trp Met Asn Pro Asn Ser Gly Asn Ala Gly
Tyr Ala1 5 1062812PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 628Arg Ala Ser Gln Ser Val Ser Ser Ser
Asn Leu Ala1 5 1062911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 629Arg Ala Ser Gln Asp Ile
Arg Asn Asp Leu Gly1 5 1063017PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 630Lys Ser Ser Gln Ser Val
Phe Tyr Ser Ser Asn Asn Lys Asn Gln Leu1 5 10
15Ala63111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 631Gln Ala Ser Gln Asp Ile Phe Lys Tyr Leu Asn1 5
1063216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 632Ser Ser Ser Gln Ser Leu Leu His Ser Asn Gly
Tyr Asn Tyr Leu Asp1 5 10 156337PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 633Trp Ala Ser Thr Arg Glu
Ser1 56347PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 634Ser Ala Ser Asn Leu Arg Ser1 5635100PRTHomo
sapiens 635Met Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His
Pro Ala1 5 10 15Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser
Gly Phe His 20 25 30Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly
Glu Arg Ile Glu 35 40 45Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys
Asp Trp Ser Phe Tyr 50 55 60Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr
Glu Lys Asp Glu Tyr Ala65 70 75 80Cys Arg Val Asn His Val Thr Leu
Ser Gln Pro Lys Ile Val Lys Trp 85 90 95Asp Arg Asp Met
1006369PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 636Asn Leu Val Pro Met Val Ala Thr Val1
563710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 637Gln Val Pro Leu Arg Pro Met Thr Ile Lys1 5
106389PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 638Gln Tyr Asp Pro Val Ala Ala Leu Phe1
56399PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 639Thr Tyr Gly Pro Val Phe Met Ser Leu1
56409PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 640Lys Tyr Thr Ser Phe Pro Trp Leu Leu1
56418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 641Arg Ala Lys Phe Lys Gln Leu Leu1
5642407PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 642Met Gly Ser His Ser Met Arg Tyr Phe Phe
Thr Ser Val Ser Arg Pro1 5 10 15Gly Arg Gly Glu Pro Arg Phe Ile Ala
Val Gly Tyr Val Asp Asp Thr 20 25 30Gln Phe Val Arg Phe Asp Ser Asp
Ala Ala Ser Gln Arg Met Glu Pro 35 40 45Arg Ala Pro Trp Ile Glu Gln
Glu Gly Pro Glu Tyr Trp Asp Gly Glu 50 55 60Thr Arg Lys Val Lys Ala
His Ser Gln Thr His Arg Val Asp Leu Gly65 70 75 80Thr Leu Arg Gly
Tyr Tyr Asn Gln Ser Glu Ala Gly Ser His Thr Val 85 90 95Gln Arg Met
Tyr Gly Cys Asp Val Gly Ser Asp Trp Arg Phe Leu Arg 100 105 110Gly
Tyr His Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Lys 115 120
125Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Thr Thr
130 135 140Lys His Lys Trp Glu Ala Ala His Val Ala Glu Gln Leu Arg
Ala Tyr145 150 155 160Leu Glu Gly Thr Cys Val Glu Trp Leu Arg Arg
Tyr Leu Glu Asn Gly 165 170 175Lys Glu Thr Leu Gln Arg Thr Asp Ala
Pro Lys Thr His Met Thr His 180 185 190His Ala Val Ser Asp His Glu
Ala Thr Leu Arg Cys Trp Ala Leu Ser 195 200 205Phe Tyr Pro Ala Glu
Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp 210 215 220Gln Thr Gln
Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly225 230 235
240Thr Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Gln Glu Gln
245 250 255Arg Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro
Leu Thr 260 265 270Leu Arg Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Met Gly Ile 275 280 285Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser
Arg His Pro Ala Glu Asn 290 295 300Gly Lys Ser Asn Phe Leu Asn Cys
Tyr Val Ser Gly Phe His Pro Ser305 310 315 320Asp Ile Glu Val Asp
Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val 325 330 335Glu His Ser
Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu 340 345 350Tyr
Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg 355 360
365Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg
370 375 380Asp Met Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala
Ile Phe385 390 395 400Pro Gly Ala Val Pro Ala Ala
40564341PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 643Arg Met Glu Pro Arg Ala Pro Trp Ile Glu
Gln Glu Gly Pro Glu Tyr1 5 10 15Trp Asp Gly Glu Thr Arg Lys Val Lys
Ala His Ser Gln Thr His Arg 20 25 30Val Asp Leu Gly Thr Leu Arg Gly
Tyr 35 4064425PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 644Ala Ala Asp Met Ala Ala Gln Thr Thr
Lys His Lys Trp Glu Ala Ala1 5 10 15His Val Ala Glu Gln Leu Arg Ala
Tyr 20 25645407PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 645Met Gly Ser His Ser Met Arg Tyr
Phe Phe Thr Ser Val Ser Arg Pro1 5 10 15Gly Arg Gly Glu Pro Arg Phe
Ile Ala Val Gly Tyr Val Asp Asp Thr 20 25 30Gln Phe Val Arg Phe Asp
Ser Asp Ala Ala Ser Gln Lys Met Glu Pro 35 40 45Arg Ala Pro Trp Ile
Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gln Glu 50 55 60Thr Arg Asn Met
Lys Ala His Ser Gln Thr Asp Arg Ala Asn Leu Gly65 70 75 80Thr Leu
Arg Gly Tyr Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Ile 85 90 95Gln
Ile Met Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Phe Leu Arg 100 105
110Gly Tyr Arg Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn
115 120 125Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln
Ile Thr 130 135 140Lys Arg Lys Trp Glu Ala Val His Ala Ala Glu Gln
Arg Arg Val Tyr145 150 155 160Leu Glu Gly Arg Cys Val Asp Gly Leu
Arg Arg Tyr Leu Glu Asn Gly 165 170 175Lys Glu Thr Leu Gln Arg Thr
Asp Pro Pro Lys Thr His Met Thr His 180 185 190His Pro Ile Ser Asp
His Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly 195 200 205Phe Tyr Pro
Ala Glu Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp 210 215 220Gln
Thr Gln Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly225 230
235 240Thr Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu
Gln 245 250 255Arg Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys
Pro Leu Thr 260 265 270Leu Arg Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Met Gly Ile 275 280 285Gln Arg Thr Pro Lys Ile Gln Val Tyr
Ser Arg His Pro Ala Glu Asn 290 295 300Gly Lys Ser Asn Phe Leu Asn
Cys Tyr Val Ser Gly Phe His Pro Ser305 310 315 320Asp Ile Glu Val
Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val 325 330 335Glu His
Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu 340 345
350Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg
355 360 365Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp
Asp Arg 370 375 380Asp Met Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Ala Ser Ser385 390 395 400Leu Pro Thr Thr Met Asn Tyr
4056464PRTHomo sapiens 646Arg Arg Val Tyr164741PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
647Lys Met Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu Gly Pro Glu Tyr1
5 10 15Trp Asp Gln Glu Thr Arg Asn Met Lys Ala His Ser Gln Thr Asp
Arg 20 25 30Ala Asn Leu Gly Thr Leu Arg Gly Tyr 35
4064825PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 648Ala Ala Asp Met Ala Ala Gln Ile Thr Lys Arg
Lys Trp Glu Ala Val1 5 10 15His Ala Ala Glu Gln Arg Arg Val Tyr 20
2564925PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 649Ala Ala Asp Thr Ala Ala Gln Ile Thr Gln Arg
Lys Trp Glu Ala Ala1 5 10 15Arg Val Ala Glu Gln Leu Arg Ala Tyr 20
256502941DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotidemodified_base(623)..(802)a, c, t, g,
unknown or othermodified_base(1463)..(1687)a, c, t, g, unknown or
other 650ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc
acagtccccg 60agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg
cgcggggtaa 120actgggaaag tgatgtcgtg tactggctcc gcctttttcc
cgagggtggg ggagaaccgt 180atataagtgc agtagtcgcc gtgaacgttc
tttttcgcaa cgggtttgcc gccagaacac 240agctgaagct tcgaggggct
cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300gccatccacg
ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg
360cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt
ccggcgctcc 420cttggagcct acctagactc agccggctct ccacgctttg
cctgaccctg cttgctcaac 480tctacgtctt tgtttcgttt tctgttctgc
gccgttacag atccaagctg tgaccggcgc 540ctactctaga gccgccacca
tggccctgcc tgtgacagcc ctgctgctgc ctctggctct 600gctgctgcat
gccgctagac ccnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 780nnnnnnnnnn nnnnnnnnnn nngaggacct
gaacaaggtg ttcccacccg aggtcgctgt 840gtttgagcca tcagaagcag
agatctccca cacccaaaag gccacactgg tgtgcctggc 900cacaggcttc
ttccccgacc acgtggagct gagctggtgg gtgaatggga aggaggtgca
960cagtggggtc tgcacggacc cgcagcccct caaggagcag cccgccctca
atgactccag 1020atactgcctg agcagccgcc tgagggtctc ggccaccttc
tggcagaacc cccgcaacca 1080cttccgctgt caagtccagt tctacgggct
ctcggagaat gacgagtgga cccaggatag 1140ggccaaaccc gtcacccaga
tcgtcagcgc cgaggcctgg ggtagagcag actgtggctt 1200tacctcggtg
tcctaccagc aaggggtcct gtctgccacc atcctctatg agatcctgct
1260agggaaggcc accctgtatg ctgtgctggt cagcgccctt gtgttgatgg
ccatggtcaa 1320gagaaaggat ttcggctccg gagccacgaa cttctctctg
ttaaagcaag caggagacgt 1380ggaagaaaac cccggtccca tggccctgcc
tgtgacagcc ctgctgctgc ctctggctct 1440gctgctgcat gccgctagac
ccnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
1560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1680nnnnnnncca aatatccaga accctgaccc
tgccgtgtac cagctgagag actctaaatc 1740cagtgacaag tctgtctgcc
tattcaccga ttttgattct caaacaaatg tgtcacaaag 1800taaggattct
gatgtgtata tcacagacaa atgcgtgcta gacatgaggt ctatggactt
1860caagagcaac agtgctgtgg cctggagcaa caaatctgac tttgcatgtg
caaacgcctt 1920caacaacagc attattccag aagacacctt cttccccagc
ccagaaagtt cctgtgatgt 1980caagctggtc gagaaaagct ttgaaacaga
tacgaaccta aactttcaaa acctgtcagt 2040gattgggttc cgaatcctcc
tcctgaaagt ggccgggttt aatctgctca tgacgctgcg 2100gctgtggtcc
agcgcggccg ctgagggcag aggaagtctt ctaacatgcg gtgacgtgga
2160ggagaatccc ggcccttccg gaatggagag cgacgagagc ggcctgcccg
ccatggagat 2220cgagtgccgc atcaccggca ccctgaacgg cgtggagttc
gagctggtgg gcggcggaga 2280gggcaccccc aagcagggcc gcatgaccaa
caagatgaag agcaccaaag gcgccctgac 2340cttcagcccc tacctgctga
gccacgtgat gggctacggc ttctaccact tcggcaccta 2400ccccagcggc
tacgagaacc ccttcctgca cgccatcaac aacggcggct acaccaacac
2460ccgcatcgag aagtacgagg acggcggcgt gctgcacgtg agcttcagct
accgctacga 2520ggccggccgc gtgatcggcg acttcaaggt ggtgggcacc
ggcttccccg aggacagcgt 2580gatcttcacc gacaagatca tccgcagcaa
cgccaccgtg gagcacctgc accccatggg 2640cgataacgtg ctggtgggca
gcttcgcccg caccttcagc ctgcgcgacg gcggctacta 2700cagcttcgtg
gtggacagcc acatgcactt caagagcgcc atccacccca gcatcctgca
2760gaacgggggc cccatgttcg ccttccgccg cgtggaggag ctgcacagca
acaccgagct 2820gggcatcgtg gagtaccagc acgccttcaa gacccccatc
gccttcgcca gatcccgcgc 2880tcagtcgtcc aattctgccg tggacggcac
cgccggaccc ggctccaccg gatctcgcta 2940g 29416513193DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
651ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc
acagtccccg 60agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg
cgcggggtaa 120actgggaaag tgatgtcgtg tactggctcc gcctttttcc
cgagggtggg ggagaaccgt 180atataagtgc agtagtcgcc gtgaacgttc
tttttcgcaa cgggtttgcc gccagaacac 240agctgaagct tcgaggggct
cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300gccatccacg
ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg
360cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt
ccggcgctcc 420cttggagcct acctagactc agccggctct ccacgctttg
cctgaccctg cttgctcaac 480tctacgtctt tgtttcgttt tctgttctgc
gccgttacag atccaagctg tgaccggcgc 540ctactctaga gccgccacca
tggccctgcc tgtgacagcc ctgctgctgc ctctggctct 600gctgctgcat
gccgctagac ccggagtctc ccagaacccc agacacaaga tcacaaagag
660gggacagaat gtaactttca ggtgtgatcc aatttctgaa cacaaccgcc
tttattggta 720ccgacagacc ctggggcagg gcccagagtt tctgacttac
ttccagaatg aagctcaact 780agaaaaatca aggctgctca gtgatcggtt
ctctgcagag aggcctaagg gatctttctc 840caccttggag atccagcgca
cagagcaggg ggactcggcc atgtatctct gtgccagcag 900cttagcgaca
gtctacgagc agtacttcgg gccgggcacc aggctcacgg tcacagagga
960cctgaacaag gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag
cagagatctc 1020ccacacccaa aaggccacac tggtgtgcct ggccacaggc
ttcttccccg accacgtgga 1080gctgagctgg tgggtgaatg ggaaggaggt
gcacagtggg gtctgcacgg acccgcagcc 1140cctcaaggag cagcccgccc
tcaatgactc cagatactgc ctgagcagcc gcctgagggt 1200ctcggccacc
ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg
1260gctctcggag aatgacgagt ggacccagga tagggccaaa cccgtcaccc
agatcgtcag 1320cgccgaggcc tggggtagag cagactgtgg ctttacctcg
gtgtcctacc agcaaggggt 1380cctgtctgcc accatcctct atgagatcct
gctagggaag gccaccctgt atgctgtgct 1440ggtcagcgcc cttgtgttga
tggccatggt caagagaaag gatttcggct ccggagccac 1500gaacttctct
ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc ccatggccct
1560gcctgtgaca gccctgctgc tgcctctggc tctgctgctg catgccgcta
gacccgagga 1620tgtggagcag agtcttttcc tgagtgtccg agagggagac
agctccgtta taaactgcac 1680ttacacagac agctcctcca cctacttata
ctggtataag caagaacctg gagcaggtct 1740ccagttgctg acgtatattt
tttcaaatat ggacatgaaa caagaccaaa gactcactgt 1800tctattgaat
aaaaaggata aacatctgtc tctgcgcatt gcagacaccc agactgggga
1860ctcagctatc tacttctgtg cagggccggg cgggtaccag aaagttacct
ttggaattgg 1920aacaaagctc caagtcatcc caaatatcca gaaccctgac
cctgccgtgt accagctgag 1980agactctaaa tccagtgaca agtctgtctg
cctattcacc gattttgatt ctcaaacaaa 2040tgtgtcacaa agtaaggatt
ctgatgtgta tatcacagac aaatgcgtgc tagacatgag 2100gtctatggac
ttcaagagca acagtgctgt ggcctggagc aacaaatctg actttgcatg
2160tgcaaacgcc ttcaacaaca gcattattcc agaagacacc ttcttcccca
gcccagaaag 2220ttcctgtgat gtcaagctgg tcgagaaaag
ctttgaaaca gatacgaacc taaactttca 2280aaacctgtca gtgattgggt
tccgaatcct cctcctgaaa gtggccgggt ttaatctgct 2340catgacgctg
cggctgtggt ccagcgcggc cgctgagggc agaggaagtc ttctaacatg
2400cggtgacgtg gaggagaatc ccggcccttc cggaatggag agcgacgaga
gcggcctgcc 2460cgccatggag atcgagtgcc gcatcaccgg caccctgaac
ggcgtggagt tcgagctggt 2520gggcggcgga gagggcaccc ccaagcaggg
ccgcatgacc aacaagatga agagcaccaa 2580aggcgccctg accttcagcc
cctacctgct gagccacgtg atgggctacg gcttctacca 2640cttcggcacc
taccccagcg gctacgagaa ccccttcctg cacgccatca acaacggcgg
2700ctacaccaac acccgcatcg agaagtacga ggacggcggc gtgctgcacg
tgagcttcag 2760ctaccgctac gaggccggcc gcgtgatcgg cgacttcaag
gtggtgggca ccggcttccc 2820cgaggacagc gtgatcttca ccgacaagat
catccgcagc aacgccaccg tggagcacct 2880gcaccccatg ggcgataacg
tgctggtggg cagcttcgcc cgcaccttca gcctgcgcga 2940cggcggctac
tacagcttcg tggtggacag ccacatgcac ttcaagagcg ccatccaccc
3000cagcatcctg cagaacgggg gccccatgtt cgccttccgc cgcgtggagg
agctgcacag 3060caacaccgag ctgggcatcg tggagtacca gcacgccttc
aagaccccca tcgccttcgc 3120cagatcccgc gctcagtcgt ccaattctgc
cgtggacggc accgccggac ccggctccac 3180cggatctcgc tag
31936523196DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 652ggatctgcga tcgctccggt gcccgtcagt
gggcagagcg cacatcgccc acagtccccg 60agaagttggg gggaggggtc ggcaattgaa
cgggtgccta gagaaggtgg cgcggggtaa 120actgggaaag tgatgtcgtg
tactggctcc gcctttttcc cgagggtggg ggagaaccgt 180atataagtgc
agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
240agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct
acctgaggcc 300gccatccacg ccggttgagt cgcgttctgc cgcctcccgc
ctgtggtgcc tcctgaactg 360cgtccgccgt ctaggtaagt ttaaagctca
ggtcgagacc gggcctttgt ccggcgctcc 420cttggagcct acctagactc
agccggctct ccacgctttg cctgaccctg cttgctcaac 480tctacgtctt
tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc
540ctactctaga gccgccacca tggccctgcc tgtgacagcc ctgctgctgc
ctctggctct 600gctgctgcat gccgctagac cccaagtgac ccagaaccca
agatacctca tcacagtgac 660tggaaagaag ttaacagtga cttgttctca
gaatatgaac catgagtata tgtcctggta 720tcgacaagac ccagggctgg
gcttaaggca gatctactat tcaatgaatg ttgaggtgac 780tgataaggga
gatgttcctg aagggtacaa agtctctcga aaagagaaga ggaatttccc
840cctgatcctg gagtcgccca gccccaacca gacctctctg tacttctgtg
ccagcagtac 900gacagggtct tcacccctcc actttgggaa cgggaccagg
ctcactgtga cagaggacct 960gaacaaggtg ttcccacccg aggtcgctgt
gtttgagcca tcagaagcag agatctccca 1020cacccaaaag gccacactgg
tgtgcctggc cacaggcttc ttccccgacc acgtggagct 1080gagctggtgg
gtgaatggga aggaggtgca cagtggggtc tgcacggacc cgcagcccct
1140caaggagcag cccgccctca atgactccag atactgcctg agcagccgcc
tgagggtctc 1200ggccaccttc tggcagaacc cccgcaacca cttccgctgt
caagtccagt tctacgggct 1260ctcggagaat gacgagtgga cccaggatag
ggccaaaccc gtcacccaga tcgtcagcgc 1320cgaggcctgg ggtagagcag
actgtggctt tacctcggtg tcctaccagc aaggggtcct 1380gtctgccacc
atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt
1440cagcgccctt gtgttgatgg ccatggtcaa gagaaaggat ttcggctccg
gagccacgaa 1500cttctctctg ttaaagcaag caggagacgt ggaagaaaac
cccggtccca tggccctgcc 1560tgtgacagcc ctgctgctgc ctctggctct
gctgctgcat gccgctagac ccaaacagga 1620ggtgacacag attcctgcag
ctctgagtgt cccagaagga gaaaacttgg ttctcaactg 1680cagtttcact
gatagcgcta tttacaacct ccagtggttt aggcaggacc ctgggaaagg
1740tctcacatct ctgttgctta ttcagtcaag tcagagagag caaacaagtg
gaagacttaa 1800tgcctcgctg gataaatcat caggacgtag tactttatac
attgcagctt ctcagcctgg 1860tgactcagcc acctacctct gtgctgttga
taactatggt cagaattttg tctttggtcc 1920cggaaccaga ttgtccgtgc
tgccaaatat ccagaaccct gaccctgccg tgtaccagct 1980gagagactct
aaatccagtg acaagtctgt ctgcctattc accgattttg attctcaaac
2040aaatgtgtca caaagtaagg attctgatgt gtatatcaca gacaaatgcg
tgctagacat 2100gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg
agcaacaaat ctgactttgc 2160atgtgcaaac gccttcaaca acagcattat
tccagaagac accttcttcc ccagcccaga 2220aagttcctgt gatgtcaagc
tggtcgagaa aagctttgaa acagatacga acctaaactt 2280tcaaaacctg
tcagtgattg ggttccgaat cctcctcctg aaagtggccg ggtttaatct
2340gctcatgacg ctgcggctgt ggtccagcgc ggccgctgag ggcagaggaa
gtcttctaac 2400atgcggtgac gtggaggaga atcccggccc ttccggaatg
gagagcgacg agagcggcct 2460gcccgccatg gagatcgagt gccgcatcac
cggcaccctg aacggcgtgg agttcgagct 2520ggtgggcggc ggagagggca
cccccaagca gggccgcatg accaacaaga tgaagagcac 2580caaaggcgcc
ctgaccttca gcccctacct gctgagccac gtgatgggct acggcttcta
2640ccacttcggc acctacccca gcggctacga gaaccccttc ctgcacgcca
tcaacaacgg 2700cggctacacc aacacccgca tcgagaagta cgaggacggc
ggcgtgctgc acgtgagctt 2760cagctaccgc tacgaggccg gccgcgtgat
cggcgacttc aaggtggtgg gcaccggctt 2820ccccgaggac agcgtgatct
tcaccgacaa gatcatccgc agcaacgcca ccgtggagca 2880cctgcacccc
atgggcgata acgtgctggt gggcagcttc gcccgcacct tcagcctgcg
2940cgacggcggc tactacagct tcgtggtgga cagccacatg cacttcaaga
gcgccatcca 3000ccccagcatc ctgcagaacg ggggccccat gttcgccttc
cgccgcgtgg aggagctgca 3060cagcaacacc gagctgggca tcgtggagta
ccagcacgcc ttcaagaccc ccatcgcctt 3120cgccagatcc cgcgctcagt
cgtccaattc tgccgtggac ggcaccgccg gacccggctc 3180caccggatct cgctag
319665310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 653Phe Pro Ala Glu Arg Asp Ile Ser Val Tyr1 5
106549PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 654Ser Pro Ala Pro Ser Leu Glu Ser Tyr1
56559PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 655Thr Ala Phe Glu Ser Ile Lys Ser Val1
56569PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 656Ala Glu Thr Ser Tyr Val Lys Val Leu1
56579PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 657Ala Leu Leu Glu Glu Glu Glu Gly Val1
56589PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 658Asp Glu Asp Gly Lys Ile Val Gly Tyr1
565911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 659Gly Leu Trp Glu Ile Glu Asn Asn Pro Thr Val1 5
10660406PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 660Met Gly Ser His Ser Met Arg Tyr Phe Phe
Thr Ser Val Ser Arg Pro1 5 10 15Gly Arg Gly Glu Pro Arg Phe Ile Ala
Val Gly Tyr Val Asp Asp Thr 20 25 30Gln Phe Val Arg Phe Asp Ser Asp
Ala Ala Ser Gln Lys Met Glu Pro 35 40 45Arg Ala Pro Trp Ile Glu Gln
Glu Gly Pro Glu Tyr Trp Asp Gln Glu 50 55 60Thr Arg Asn Met Lys Ala
His Ser Gln Thr Asp Arg Ala Asn Leu Gly65 70 75 80Thr Leu Arg Gly
Tyr Tyr Asn Gln Ser Glu Asp Gly Ser His Thr Ile 85 90 95Gln Ile Met
Tyr Gly Cys Asp Val Gly Pro Asp Gly Arg Phe Leu Arg 100 105 110Gly
Tyr Arg Gln Asp Ala Tyr Asp Gly Lys Asp Tyr Ile Ala Leu Asn 115 120
125Glu Asp Leu Arg Ser Trp Thr Ala Ala Asp Met Ala Ala Gln Ile Thr
130 135 140Lys Arg Lys Trp Glu Ala Val His Ala Ala Glu Gln Arg Arg
Val Tyr145 150 155 160Leu Glu Gly Arg Cys Val Asp Gly Leu Arg Arg
Tyr Leu Glu Asn Gly 165 170 175Lys Glu Thr Leu Gln Arg Thr Asp Pro
Pro Lys Thr His Met Thr His 180 185 190His Pro Ile Ser Asp His Glu
Ala Thr Leu Arg Cys Trp Ala Leu Gly 195 200 205Phe Tyr Pro Ala Glu
Ile Thr Leu Thr Trp Gln Arg Asp Gly Glu Asp 210 215 220Gln Thr Gln
Asp Thr Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly225 230 235
240Thr Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln
245 250 255Arg Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Lys Pro
Leu Thr 260 265 270Leu Arg Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Met Gly Ile 275 280 285Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser
Arg His Pro Ala Glu Asn 290 295 300Gly Lys Ser Asn Phe Leu Asn Cys
Tyr Val Ser Gly Phe His Pro Ser305 310 315 320Asp Ile Glu Val Asp
Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val 325 330 335Glu His Ser
Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu 340 345 350Tyr
Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg 355 360
365Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg
370 375 380Asp Met Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asn
Thr Asp385 390 395 400Asn Asn Leu Ala Val Tyr 40566136PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
661Trp Ile Glu Gln Glu Gly Pro Glu Tyr Trp Asp Gln Glu Thr Arg Asn1
5 10 15Met Lys Ala His Ser Gln Thr Asp Arg Ala Asn Leu Gly Thr Leu
Arg 20 25 30Gly Tyr Tyr Asn 3566227PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 662Asp
Met Ala Ala Gln Ile Thr Lys Arg Lys Trp Glu Ala Val His Ala1 5 10
15Ala Glu Gln Arg Arg Val Tyr Leu Glu Gly Arg 20
2566312PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 663Thr Asp Arg Ala Asn Leu Gly Thr Leu Arg Gly
Tyr1 5 106649PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 664Leu Arg Ser Trp Thr Ala Ala Asp Met1
566541PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 665Arg Thr Glu Pro Arg Ala Pro Trp Ile Glu
Gln Glu Gly Pro Glu Tyr1 5 10 15Trp Asp Arg Asn Thr Gln Ile Phe Lys
Thr Asn Thr Gln Thr Tyr Arg 20 25 30Glu Ser Leu Arg Asn Leu Arg Gly
Tyr 35 40
* * * * *
References